Tideglusib Rescues Neurite Pathology of SPG11 iPSC Derived Cortical Neurons

Mutations in SPG11 cause a complicated autosomal recessive form of hereditary spastic paraplegia (HSP). Mechanistically, there are indications for the dysregulation of the GSK3β/βCat signaling pathway in SPG11. In this study, we tested the therapeutic potential of the GSK3β inhibitor, tideglusib, to rescue neurodegeneration associated characteristics in an induced pluripotent stem cells (iPSCs) derived neuronal model from SPG11 patients and matched healthy controls as well as a CRISPR-Cas9 mediated SPG11 knock-out line and respective control. SPG11-iPSC derived cortical neurons, as well as the genome edited neurons exhibited shorter and less complex neurites than controls. Administration of tideglusib to these lines led to the rescue of neuritic impairments. Moreover, the treatment restored increased cell death and ameliorated the membranous inclusions in iPSC derived SPG11 neurons. Our results provide a first evidence for the rescue of neurite pathology in SPG11-HSP by tideglusib. The current lack of disease-modifying treatments for SPG11 and related types of complicated HSP renders tideglusib a candidate compound for future clinical application

Generation and characterization of an endogenously tagged SPG11-human iPSC line by CRISPR/Cas9 mediated knock-in

Pathogenic bi-allelic variants in the SPG11 gene result in rare motor neuron disorders such as Hereditary Spastic Paraplegia type 11, Charcot-Marie Tooth, and Juvenile Amyotrophic Lateral Sclerosis-5. The main challenge in SPG11-linked disease research is the lack of antibodies against SPG11 encoded spatacsin. Here, we describe the CRISPR/Cas9 mediated generation and validation of an endogenously tagged SPG11- human iPSC line that contains an HA tag at the C-terminus of SPG11. The line exhibits multi-lineage differentiation potential and holds promise for studying the role of spatacsin and for the elucidation of SPG11-associated pathogenesis

Pathogenic SCN2A variants cause early-stage dysfunction in patient-derived neurons

Pathogenic heterozygous variants in SCN2A, which encodes the neuronal sodium channel NaV1.2, cause different types of epilepsy or intellectual disability (ID)/autism without seizures. Previous studies using mouse models or heterologous systems suggest that NaV1.2 channel gain-of-function typically causes epilepsy, whereas loss-of-function leads to ID/autism. How altered channel biophysics translate into patient neurons remains unknown. Here, we investigated iPSC-derived early-stage cortical neurons from ID patients harboring diverse pathogenic SCN2A variants [p.(Leu611Valfs*35); p.(Arg937Cys); p.(Trp1716*)], and compared them to neurons from an epileptic encephalopathy patient [p.(Glu1803Gly)] and controls. ID neurons consistently expressed lower NaV1.2 protein levels. In neurons with the frameshift variant, NaV1.2 mRNA and protein levels were reduced by ~ 50%, suggesting nonsense-mediated decay and haploinsufficiency. In other ID neurons, only protein levels were reduced implying NaV1.2 instability. Electrophysiological analysis revealed decreased sodium current density and impaired action potential (AP) firing in ID neurons, consistent with reduced NaV1.2 levels. By contrast, epilepsy neurons displayed no change in NaV1.2 levels or sodium current density, but impaired sodium channel inactivation. Single-cell transcriptomics identified dysregulation of distinct molecular pathways including inhibition of oxidative phosphorylation in neurons with SCN2A haploinsufficiency, and activation of calcium signaling and neurotransmission in epilepsy neurons. Together, our patient iPSC-derived neurons reveal characteristic sodium channel dysfunction consistent with biophysical changes previously observed in heterologous systems. Additionally, our model links the channel dysfunction in ID to reduced NaV1.2 levels and uncovers impaired AP firing in early-stage neurons. The altered molecular pathways may reflect a homeostatic response to NaV1.2 dysfunction and can guide further investigations

Pathogenic SCN2A variants cause early-stage dysfunction in patient-derived neurons

Pathogenic heterozygous variants in SCN2A, which encodes the neuronal sodium channel NaV1.2, cause different types of epilepsy or intellectual disability (ID)/autism without seizures. Previous studies using mouse models or heterologous systems suggest that NaV1.2 channel gain-of-function typically causes epilepsy, whereas loss-of-function leads to ID/autism. How altered channel biophysics translate into patient neurons remains unknown. Here, we investigated iPSC-derived early-stage cortical neurons from ID patients harboring diverse pathogenic SCN2A variants [p.(Leu611Valfs*35); p.(Arg937Cys); p.(Trp1716*)], and compared them to neurons from an epileptic encephalopathy patient [p.(Glu1803Gly)] and controls. ID neurons consistently expressed lower NaV1.2 protein levels. In neurons with the frameshift variant, NaV1.2 mRNA and protein levels were reduced by ~ 50%, suggesting nonsense-mediated decay and haploinsufficiency. In other ID neurons, only protein levels were reduced implying NaV1.2 instability. Electrophysiological analysis revealed decreased sodium current density and impaired action potential (AP) firing in ID neurons, consistent with reduced NaV1.2 levels. By contrast, epilepsy neurons displayed no change in NaV1.2 levels or sodium current density, but impaired sodium channel inactivation. Single-cell transcriptomics identified dysregulation of distinct molecular pathways including inhibition of oxidative phosphorylation in neurons with SCN2A haploinsufficiency, and activation of calcium signaling and neurotransmission in epilepsy neurons. Together, our patient iPSC-derived neurons reveal characteristic sodium channel dysfunction consistent with biophysical changes previously observed in heterologous systems. Additionally, our model links the channel dysfunction in ID to reduced NaV1.2 levels and uncovers impaired AP firing in early-stage neurons. The altered molecular pathways may reflect a homeostatic response to NaV1.2 dysfunction and can guide further investigations

Tideglusib Rescues Neurite Pathology of SPG11 iPSC Derived Cortical Neurons

Mutations in SPG11 cause a complicated autosomal recessive form of hereditary spastic paraplegia (HSP). Mechanistically, there are indications for the dysregulation of the GSK3β/βCat signaling pathway in SPG11. In this study, we tested the therapeutic potential of the GSK3β inhibitor, tideglusib, to rescue neurodegeneration associated characteristics in an induced pluripotent stem cells (iPSCs) derived neuronal model from SPG11 patients and matched healthy controls as well as a CRISPR-Cas9 mediated SPG11 knock-out line and respective control. SPG11-iPSC derived cortical neurons, as well as the genome edited neurons exhibited shorter and less complex neurites than controls. Administration of tideglusib to these lines led to the rescue of neuritic impairments. Moreover, the treatment restored increased cell death and ameliorated the membranous inclusions in iPSC derived SPG11 neurons. Our results provide a first evidence for the rescue of neurite pathology in SPG11-HSP by tideglusib. The current lack of disease-modifying treatments for SPG11 and related types of complicated HSP renders tideglusib a candidate compound for future clinical application

Store-operated calcium entry is reduced in spastin-linked hereditary spastic paraplegia

Pathogenic variants in SPAST, the gene coding for spastin, are the single most common cause of hereditary spastic paraplegia, a progressive motor neuron disease. Spastin regulates key cellular functions, including microtubule-severing and endoplasmic reticulum-morphogenesis. However, it remains unclear how alterations in these cellular functions due to SPAST pathogenic variants result in motor neuron dysfunction. Since spastin influences both microtubule network and endoplasmic reticulum structure, we hypothesized that spastin is necessary for the regulation of Ca2+ homeostasis via store-operated calcium entry. Here, we show that the lack of spastin enlarges the endoplasmic reticulum and reduces store-operated calcium entry. In addition, elevated levels of different spastin variants induced clustering of STIM1 within the endoplasmic reticulum, altered the transport of STIM1 to the plasma membrane and reduced store-operated calcium entry, which could be rescued by exogenous expression of STIM1. Importantly, store-operated calcium entry was strongly reduced in induced pluripotent stem cell-derived neurons from hereditary spastic paraplegia patients with pathogenic variants in SPAST resulting in spastin haploinsufficiency. These neurons developed axonal swellings in response to lack of spastin. We were able to rescue both store-operated calcium entry and axonal swellings in SPAST patient neurons by restoring spastin levels, using CRISPR/Cas9 to correct the pathogenic variants in SPAST. These findings demonstrate that proper amounts of spastin are a key regulatory component for store-operated calcium entry mediated Ca2+ homeostasis and suggest store-operated calcium entry as a disease relevant mechanism of spastin-linked motor neuron disease

Pathogenic SCN2A variants cause early-stage dysfunction in patient-derived neurons

Pathogenic heterozygous variants in SCN2A, which encodes the neuronal sodium channel Na(V)1.2, cause different types of epilepsy or intellectual disability (ID)/autism without seizures. Previous studies using mouse models or heterologous systems suggest that Na(V)1.2 channel gain-of-function typically causes epilepsy, whereas loss-of-function leads to ID/autism. How altered channel biophysics translate into patient neurons remains unknown. Here, we investigated iPSC-derived early-stage cortical neurons from ID patients harboring diverse pathogenic SCN2A variants [p.(Leu611Valfs*35); p.(Arg937Cys); p.(Trp1716*)] and compared them with neurons from an epileptic encephalopathy (EE) patient [p.(Glu1803Gly)] and controls. ID neurons consistently expressed lower Na(V)1.2 protein levels. In neurons with the frameshift variant, Na(V)1.2 mRNA and protein levels were reduced by similar to 50%, suggesting nonsense-mediated decay and haploinsufficiency. In other ID neurons, only protein levels were reduced implying Na(V)1.2 instability. Electrophysiological analysis revealed decreased sodium current density and impaired action potential (AP) firing in ID neurons, consistent with reduced Na(V)1.2 levels. In contrast, epilepsy neurons displayed no change in Na(V)1.2 levels or sodium current density, but impaired sodium channel inactivation. Single-cell transcriptomics identified dysregulation of distinct molecular pathways including inhibition of oxidative phosphorylation in neurons with SCN2A haploinsufficiency and activation of calcium signaling and neurotransmission in epilepsy neurons. Together, our patient iPSC-derived neurons reveal characteristic sodium channel dysfunction consistent with biophysical changes previously observed in heterologous systems. Additionally, our model links the channel dysfunction in ID to reduced Na(V)1.2 levels and uncovers impaired AP firing in early-stage neurons. The altered molecular pathways may reflect a homeostatic response to Na(V)1.2 dysfunction and can guide further investigations.ISSN:0964-6906ISSN:1460-208