Hereditary Spastic Paraplegias (HSPs) are a group of heterogeneous neurological diseases characterized by axonal degeneration of corticospinal motor neurons. SPG15, a common autosomal recessive form of HSP, is caused by mutations in the ZFYVE26 gene that encodes the spastizin protein. Spastizin partially localizes to mitochondria, suggesting a potential role in mitochondrial function. To investigate this possibility and explore treatments to mitigate neurodegeneration caused by spastizin deficiency, SPG15 patient-specific induced pluripotent stem cells (iPSCs) were generated from patient fibroblasts and differentiated into cortical projection neurons. The SPG15 iPSC-derived neurons exhibited reduced ATP production compared to control neurons, indicating mitochondrial dysfunction in spastizin-deficient neurons. Also, given increased fragmentation of mitochondria in SPG15 neurons, we examined whether restoring mitochondrial morphology dynamics using P110, a peptide inhibitor of the mitochondrial fission protein DRP1, could protect SPG15 neurons. Indeed, treatment with P110 significantly suppressed the accumulated axonal swellings in SPG15 neurons. Further examination of the underlying mechanisms revealed that P110 restored mitochondrial morphology, ameliorated mitochondrial oxidative stress, and improved mitochondrial health, but it did not affect necrosis-related membrane integrity, suggesting specific targeting of mitochondrial deficits by P110. Furthermore, P110 significantly mitigated disease-related apoptosis in SPG15 neurons. Collectively, these findings reveal that restoring mitochondrial morphology and function using P110 lessens mitochondrial oxidative stress and mitigates degeneration of SPG15 neurons, offering a novel therapeutic approach for SPG15.

Hereditary spastic paraplegias (HSPs) comprise a large, heterogeneous group of inherited disorders characterized by length-dependent axonal degeneration of corticospinal motor neurons, leading to lower extremity spasticity and gait impairment. Currently, there are no effective treatments for HSPs targeting axonal dysfunction. Our previous study showed that lipid defects in glial cells result in degeneration of iPSC-derived cortical projection neurons (PNs) in SPG3A, the most common early-onset form of HSP caused by autosomal dominant mutations in the ATL1 gene encoding atlastin-1. However, how cortical PNs degenerate and whether therapeutic compounds targeting lipid defects can effectively mitigate degeneration in human ATL1 neurons remain unclear. Here, by comparing SPG3A patient iPSC-derived neurons with control cells using RNA-sequencing, we identified synaptic dysfunction as a top-altered pathway in addition to lipid-related pathways. To examine the novel role of synaptic dysfunction in SPG3A, we generated patient-specific iPSCs from two SPG3A patients with distinct missense mutations and differentiated them into cortical PNs. We observed significant reductions of synaptic genes and proteins in cortical PNs from both SPG3A-P342S and SPG3A-M408T patient iPSCs, emphasizing synaptic dysfunction in SPG3A neurons. Calcium imaging revealed a significant reduction of activity in SPG3A cortical neurons compared to control neurons, further supporting functional deficits in SPG3A neurons. To further examine the role of these processes in HSP pathogenesis, we treated cells with LXR623, an orally bioavailable liver-X-receptor (LXR) agonist that can modulate lipid metabolism and transfer. LXR623 significantly mitigated the reduction in synaptic proteins and calcium activity and rescued axonal degeneration and apoptosis in SPG3A cortical PNs. Furthermore, analyses of lipid and synaptic genes and proteins revealed that LXR623 treatment effectively restored mRNA expression patterns for these pathways in SPG3A neurons. Taken together, our data demonstrate the role of synaptic dysfunction in degeneration of SPG3A neurons and highlight the therapeutic potential of an LXR agonist in mitigating human cortical neuron degeneration in HSP.

>Axonal degeneration underlies many debilitating diseases including hereditary spastic paraplegia(HSP),a genetically and clinically diverse group of disorders characterized by spasticity and weakness of the lower extremities.HSP is one significant cause of chronic neurodisability due to the lack of effective treatments and a wide range of onset ages from early childhood to 70 years.

Background Biallelic mutations in CYP27A1 and CYP7B1 , two critical genes regulating cholesterol and bile acid metabolism, cause cerebrotendinous xanthomatosis (CTX) and hereditary spastic paraplegia type 5 (SPG5), respectively. These rare diseases are characterized by progressive degeneration of corticospinal motor neuron axons, yet the underlying pathogenic mechanisms and strategies to mitigate axonal degeneration remain elusive. Methods To generate induced pluripotent stem cell (iPSC)-based models for CTX and SPG5, we reprogrammed patient skin fibroblasts into iPSCs by transducing fibroblast cells with episomal vectors containing pluripotency factors. These patient-specific iPSCs, as well as control iPSCs, were differentiated into cortical projection neurons (PNs) and examined for biochemical alterations and disease-related phenotypes. Results CTX and SPG5 patient iPSC-derived cortical PNs recapitulated several disease-specific biochemical changes and axonal defects of both diseases. Notably, the bile acid chenodeoxycholic acid (CDCA) effectively mitigated the biochemical alterations and rescued axonal degeneration in patient iPSC-derived neurons. To further examine underlying disease mechanisms, we developed CYP7B1 knockout human embryonic stem cell (hESC) lines using CRISPR-cas9-mediated gene editing and, following differentiation, examined hESC-derived cortical PNs. Knockout of CYP7B1 resulted in similar axonal vesiculation and degeneration in human cortical PN axons, confirming a cause-effect relationship between gene deficiency and axonal degeneration. Interestingly, CYP7B1 deficiency led to impaired neurofilament expression and organization as well as axonal degeneration, which could be rescued with CDCA, establishing a new disease mechanism and therapeutic target to mitigate axonal degeneration. Conclusions Our data demonstrate disease-specific lipid disturbances and axonopathy mechanisms in human pluripotent stem cell-based neuronal models of CTX and SPG5 and identify CDCA, an established treatment of CTX, as a potential pharmacotherapy for SPG5. We propose this novel treatment strategy to rescue axonal degeneration in SPG5, a currently incurable condition.

Spastic paraplegia type 11 (SPG11) is a common autosomal recessive form of hereditary spastic paraplegia (HSP) characterized by the degeneration of cortical motor neuron axons, leading to muscle spasticity and weakness. Impaired lipid trafficking is an emerging pathology in neurodegenerative diseases including SPG11, though its role in axonal degeneration of human SPG11 neurons remains unknown. Here, we established a pluripotent stem cell-based SPG11 model by knocking down the SPG11 gene in human embryonic stem cells (hESCs). These stem cells were then differentiated into cortical projection neurons (PNs), the cell types affected in HSP patients, to examine axonal defects and cholesterol distributions. Our data revealed that SPG11 deficiency led to reduced axonal outgrowth, impaired axonal transport, and accumulated swellings, recapitulating disease-specific phenotypes. In SPG11-knockdown neurons, cholesterol was accumulated in lysosome and reduced in plasma membrane, revealing impairments in cholesterol trafficking. Strikingly, the liver-X-receptor (LXR) agonists restored cholesterol homeostasis, leading to the rescue of subsequent axonal defects in SPG11-deficient cortical PNs. To further determine the implication of impaired cholesterol homeostasis in SPG11, we examined the cholesterol distribution in cortical PNs generated from SPG11 disease-mutation knock-in hESCs, and observed a similar cholesterol trafficking impairment. Moreover, LXR agonists rescued the aberrant cholesterol distribution and mitigated the degeneration of SPG11 disease-mutated neurons. Taken together, our data demonstrate impaired cholesterol trafficking underlying axonal degeneration of SPG11 human neurons, and highlight the therapeutic potential of LXR agonists for SPG11 through restoring cholesterol homeostasis.

Hereditary spastic paraplegias (HSPs), a heterogeneous group of neurological disorders, are caused by axonal degeneration involving the long corticospinal tract which leads to the progressive spasticity and weakness of lower limbs. Over 80 different gene loci associated with HSP have been identified. Studying various HSP proteins reveals several common cellular themes in these debilitating diseases including vesicular trafficking, organelle shaping and morphogenesis, lipid/cholesterol metabolism, and endolysosomal and autophagic dysfunction. Though animal and cell models have been developed to study HSP, it remains a challenge to obtain patient-specific cortical neurons that are specifically affected and impaired in HSP. Recently, the discovery of induced pluripotent stem cells (iPSCs) provides a promising tool in neurological disease modeling due to their ability of self-renewal and capability of differentiation into various cell types. HSP patient iPSC-derived cortical projection neurons offer a unique source for HSP modeling to understand the molecular and cellular mechanisms, reveal neuronal pathological phenotypes, and test potential drugs. Here, we will introduce HSP and different common cellular themes, summarize in vitro HSP models using human pluripotent stem cells, and discuss the current challenges and future directions of HSP iPSC modeling.

Increases in blood concentrations of glucose induced by epinephrine, of lactate induced by epinephrine and isoproterenol, and of glycerol and insulin induced by isoproterenol in rats showed graded responses dependent on the dose of the catecholamines and were blocked also progressively by increasing doses of 5-(3-tert-butylamino-2-hydroxy)propoxy-3,4-dihydrocarbostyril hydrochloride (carteolol), a new beta-adrenergic blocking agent. The ed50 estimated for these metabolic parameters, other than blood insulin, was 100–200 μg/kg of body wt for epinephrine and 10–20 μg/kg for isoproterenol, while the “corrected id50” for carteolol, reflecting the dissociation constant of the antagonist-receptor complex, was 1–3 μg/kg. It is concluded that the beta-adrenergic receptors of the same character concerning the affinity to agonists and antagonists mediate the increase of blood levels of these carbohydrate and lipid intermediary metabolites, and that the affinity of carteolol is one order higher than that of isoproterenol, which in turn is ten times higher than that of epinephrine. In contrast, much higher ed50 for isoproterenol and “corrected id50” for carteolol were obtained when hyperinsulinemia was used as a measure of beta action, suggesting that the beta-adrenergic receptor mediating pancreatic secretion of insulin is distinct in nature from the receptors involved in the control of carbohydrate and lipid metabolism. Comparison of the potency of carteolol with those of propranolol and pindolol showed that carteolol is the most potent beta-adrenergic blocking agent.

Axonal degeneration underlies many debilitating diseases including hereditary spastic paraplegias (HSPs). HSPs are a large heterogeneous group of neurodegenerative diseases characterized by axonopathy involving the long corticospinal tract. How axons of these cortical projection neurons specifically degenerate in HSPs remains largely unclear partially due to the lack of human models to monitor the dynamic process of axonal degeneration. With the development of induced pluripotent stem cell (iPSC) technology, patient-specific iPSCs are successfully generated from HSP patients, providing a unique paradigm to study the axonal degeneration in patient-derived neurons in live cultures. In this chapter, we will summarize the procedures to examine axonal defects in iPSC models of HSPs and discuss the challenges and future applications in order to rescue axonal degeneration in HSPs.

Motor neurons are large projection neurons classified into upper and lower motor neurons responsible for controlling the movement of muscles. Degeneration of motor neurons results in progressive muscle weakness, which underlies several debilitating neurological disorders including amyotrophic lateral sclerosis (ALS), hereditary spastic paraplegias (HSP), and spinal muscular atrophy (SMA). With the development of induced pluripotent stem cell (iPSC) technology, human iPSCs can be derived from patients and further differentiated into motor neurons. Motor neuron disease models can also be generated by genetically modifying human pluripotent stem cells. The efficiency of gene targeting in human cells had been very low, but is greatly improved with recent gene editing technologies such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and CRISPR-Cas9. The combination of human stem cell-based models and gene editing tools provides unique paradigms to dissect pathogenic mechanisms and to explore therapeutics for these devastating diseases. Owing to the critical role of several genes in the etiology of motor neuron diseases, targeted gene therapies have been developed, including antisense oligonucleotides, viral-based gene delivery, and in situ gene editing. This review summarizes recent advancements in these areas and discusses future challenges toward the development of transformative medicines for motor neuron diseases.

Understanding the biological processes that determine the entry of three germ layers of human pluripotent stem cells (hPSCs) is a central question in developmental and stem cell biology. Here, we genetically engineered hPSCs with the germ layer reporter and inducible CRISPR/Cas9 knockout system, and a genome-scale screening was performed to define pathways restricting germ layer specification. Genes clustered in the key biological processes, including embryonic development, mRNA processing, metabolism, and epigenetic regulation, were centered in the governance of pluripotency and lineage development. Other than typical pluripotent transcription factors and signaling molecules, loss of function of mesendodermal specifiers resulted in advanced neuroectodermal differentiation, given their inter-germ layer antagonizing effect. Regarding the epigenetic superfamily, microRNAs enriched in hPSCs showed clear germ layer-targeting specificity. The cholesterol synthesis pathway maintained hPSCs via retardation of neuroectoderm specification. Thus, in this study, we identified a full landscape of genetic wiring and biological processes that control hPSC self-renewal and trilineage specification.

Charcot-Marie-Tooth (CMT) disease is one of the most common genetically inherited neurological disorders and CMT type 2A (CMT 2A) is caused by dominant mutations in the mitofusin-2 (MFN2) gene. MFN2 is located in the outer mitochondrial membrane and is a mediator of mitochondrial fusion, with an essential role in maintaining normal neuronal functions. Although loss of MFN2 induces axonal neuropathy, the detailed mechanism by which MFN2 deficiency results in axonal degeneration of human spinal motor neurons remains largely unknown. In this study, we generated MFN2-knockdown human embryonic stem cell (hESC) lines using lentivirus expressing MFN2 short hairpin RNA (shRNA). Using these hESC lines, we found that MFN2 loss did not affect spinal motor neuron differentiation from hESCs but resulted in mitochondrial fragmentation and dysfunction as determined by live-cell imaging. Notably, MFN2-knockodwn spinal motor neurons exhibited CMT2A disease-related phenotypes, including extensive perikaryal inclusions of phosphorylated neurofilament heavy chain (pNfH), frequent axonal swellings, and increased pNfH levels in long-term cultures. Importantly, MFN2 deficit impaired anterograde and retrograde mitochondrial transport within axons, and reduced the mRNA and protein levels of kinesin and dynein, indicating the interfered motor protein expression induced by MFN2 deficiency. Our results reveal that MFN2 knockdown induced axonal degeneration of spinal motor neurons and defects in mitochondrial morphology and function. The impaired mitochondrial transport in MFN2-knockdown spinal motor neurons is mediated, at least partially, by the altered motor proteins, providing potential therapeutic targets for rescuing axonal degeneration of spinal motor neurons in CMT2A disease.

Allogeneic immune rejection is a major barrier for the application of human pluripotent stem cells (hPSCs) in regenerative medicine. A broad spectrum of immune cells, including T cells, natural killer (NK) cells, and antigen-presenting cells, which either cause direct cell killing or constitute an immunogenic environment, are involved in allograft immune rejection. A strategy to protect donor cells from cytotoxicity while decreasing the secretion of inflammatory cytokines of lymphocytes is still lacking. Here, we engineered hPSCs with no surface expression of classical human leukocyte antigen (HLA) class I proteins via beta-2 microglobulin (B2M) knockout or biallelic knockin of HLA-G1 within the frame of endogenous B2M loci. Elimination of the surface expression of HLA class I proteins protected the engineered hPSCs from cytotoxicity mediated by T cells. However, this lack of surface expression also resulted in missing-self response and NK cell activation, which were largely compromised by expression of β2m-HLA-G1 fusion proteins. We also proved that the engineered β2m-HLA-G5 fusion proteins were soluble, secretable, and capable of safeguarding low immunogenic environments by lowering inflammatory cytokines secretion in allografts. Our current study reveals a novel strategy that may offer unique advantages to construct hypoimmunogenic hPSCs via the expression of membrane-bound and secreted β2m-HLA-G fusion proteins. These engineered hPSCs are expected to serve as an unlimited cell source for generating universally compatible "off-the-shelf" cell grafts in the future.

Medial ganglionic eminence (MGE)-like cells yielded from human pluripotent stem cells (hPSCs) hold great potentials for cell therapies of related neurological disorders. However, cues that orchestrate the maintenance versus differentiation of human MGE progenitors, and ways for large-scale expansion of these cells have not been investigated. Here, we report that WNT/CTNNB1 signaling plays an essential role in maintaining MGE-like cells derived from hPSCs. Ablation of CTNNB1 in MGE cells led to precocious cell-cycle exit and advanced neuronal differentiation. Activation of WNT signaling through genetic or chemical approach was sufficient to maintain MGE cells in an expandable manner with authentic neuronal differentiation potencies through activation of endogenous NOTCH signaling. Our findings reveal that WNT/NOTCH signaling cascade is a key player in governing the maintenance versus terminal differentiation of MGE progenitors in humans. Large-scale expansion of functional MGE progenitors for cell therapies can therefore be achieved by modifying WNT/NOTCH pathway.

It remains largely unknown how Zika virus (ZIKV) infection causes severe microcephaly in human newborns. We examined an Asian lineage ZIKV, SZ01, which similarly infected and demonstrated comparable growth arrest and apoptotic pathological changes in human neuroprogenitors (NPCs) from forebrain dorsal, forebrain ventral as well as hindbrain and spinal cord brain organoids derived from human pluripotent stem cells. Transcriptome profiling showed common overactivated antiviral response in all regional NPCs upon ZIKV infection. ZIKV infection directly activated a subset of IFN-stimulated genes (ISGs) in human NPCs, which depended on the presence of IRF3 and NF-κB rather than IFN production and secretion, highlighting a key role of IFN-independent acute antiviral pathway underlying ZIKV infection-caused neuropathy. Our findings therefore reveal that overactivated antiviral response is detrimental rather than protective in human NPCs, and the IFN-independent acute antiviral pathway may serve as a potential target to ameliorate ZIKV infection-triggered neuropathy.

Most of the brain tumors are malignant with an extremely poor survival rate. Recent progress in identifying cancer stem cells (CSCs) within the brain tumors is starting to revolutionize our understanding in the imitation and progression of tumors as well as relapse and the development of therapeutic strategies. Suspension spheroid body culture paradigm is a routine method in enriching CSCs. While, it was reported recently that CSCs within the brain tumor may also be enriched through adherent monolayer culture with optimized properties. In the present study, 18 surgically resected brain tumors were used for analyzing the feasibility of adherent enrichment of CSCs. The results indicated that 50% of glioblastomas were able to generate adherent CSCs, which were uniformly positive for Sox2, CD133, GFAP and Nestin. However, adherent culture paradigm failed to yield CSCs in secondary brain tumors, including neurocytomas, ependymomas, germ cell tumors or low-grade astrocytomas, which is most likely due to a lack of CD133+/Sox2+ cells within the original biopsies. Therefore, it was concluded that the adherent culture paradigm may serve as a reliable method in enriching brain CSCs, but this method is more suitable for enriching CD133+/Sox2+ CSCs in glioblastomas.

It is highly desirable to specify human developmental principles in an appropriate human model with advanced genetic tools. However, genetically engineering human cells with lineage-tracing systems has not been achieved. Here we introduce strategies to construct lineage-tracing systems in human embryonic stem cells (hESCs). The AAVS1 locus was suitable for the integration of the conditional reporter. The Cre-LoxP and Flp-FRT systems were highly sensitive, which may cause inaccurate lineage labeling in human cells. The recombination sensitivity and tracing fidelity could be finely tuned by modification of the LoxP recombination site. Moreover, tamoxifen-controllable CreERT2-LoxP and FlpERT2-FRT systems showed compelling advantages in tightly tracing human lineages temporally. In proof-of-principle experiments, we traced human PAX6+ neuroectoderm cells and revealed their full neural lineage differentiation potency both in vitro and in vivo. Devising and optimizing of lineage-tracing systems in hESCs will thus set up a solid foundation for human developmental studies.

Neuroectoderm is an important neural precursor. However, chromatin remodeling and its epigenetic regulatory roles during the differentiation of human neuroectodermal cells (hNECs) from human embryonic stem cells (hESCs) remain largely unexplored. Here, we obtained hNECs through directed differentiation from hESCs, and determined chromatin states in the two cell types. Upon differentiation, H2A.Z-mediated nucleosome depletion leads to an open chromatin structure in promoters and upregulates expression of neuroectodermal genes. Increase in H3K9ac signals and decrease in H3K27me3 signals in promoters result in an active chromatin state and activate neuroectodermal genes. Conversely, decrease in H3K9ac signals and increase in H3K27me3 signals in promoters repress pluripotency genes. Moreover, H3K9ac signals facilitate the pluripotency factor Sox2 binding to target sites unique to hNECs. Knockdown of the acetyltransferase Kat2b erases H3K9ac signals, disrupts Sox2 binding, and fails the differentiation. Our results demonstrate a hierarchy of epigenetic regulation of gene expression during the differentiation of hNECs from hESCs through chromatin remodeling.