Deficiency of the Lysosomal Protein CLN5 Alters Lysosomal Function and Movement

Batten disease is a devastating, childhood, rare neurodegenerative disease characterised by the rapid deterioration of cognition and movement, leading to death within ten to thirty years of age. One of the thirteen Batten disease forms, CLN5 Batten disease, is caused by mutations in the CLN5 gene, leading to motor deficits, mental deterioration, cognitive impairment, visual impairment, and epileptic seizures in children. A characteristic pathology in CLN5 Batten disease is the defects in lysosomes, leading to neuronal dysfunction. In this study, we aimed to investigate the lysosomal changes in CLN5-deficient human neurons. We used an induced pluripotent stem cell system, which generates pure human cortical-like glutamatergic neurons. Using CRISPRi, we inhibited the expression of CLN5 in human neurons. The CLN5-deficient human neurons showed reduced acidic organelles and reduced lysosomal enzyme activity measured by microscopy and flow cytometry. Furthermore, the CLN5-deficient human neurons also showed impaired lysosomal movement—a phenotype that has never been reported in CLN5 Batten disease. Lysosomal trafficking is key to maintain local degradation of cellular wastes, especially in long neuronal projections, and our results from the human neuronal model present a key finding to understand the underlying lysosomal pathology in neurodegenerative diseases.

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Deficiency of the lysosomal protein CLN5 alters lysosomal function and movement

10.1101/2021.08.24.457390 ◽

2021 ◽

Author(s):

Indranil Basak ◽

Rachel A Hansen ◽

Michael E Ward ◽

Stephanie M Hughes

Keyword(s):

Batten Disease ◽

Induced Pluripotent Stem Cell ◽

Epileptic Seizures ◽

Neuronal Model ◽

Cell System ◽

Motor Deficits ◽

Glutamatergic Neurons ◽

Human Neurons ◽

Stem Cell System ◽

Induced Pluripotent

Batten disease is a devastating childhood rare neurodegenerative disease characterized by rapid deterioration of cognition and movement, leading to death within ten to thirty years of age. One of the thirteen Batten disease forms, CLN5 Batten disease, is caused by mutations in the CLN5 gene leading to motor deficits, mental deterioration, cognitive impairment, visual impairment, and epileptic seizures in children. A characteristic pathology in CLN5 Batten disease is the defects in lysosomes, leading to neuronal dysfunction. In this study, we aimed to investigate the lysosomal changes in CLN5-deficient human neurons. We used an induced pluripotent stem cell system, which generates pure human cortical-like glutamatergic neurons. Using CRISPRi, we inhibited the expression of CLN5 in human neurons. The CLN5-deficient human neurons showed neutralised lysosomal acidity and reduced lysosomal enzyme activity measured by microscopy and flow cytometry. Furthermore, the CLN5-deficient human neurons also showed impaired lysosomal movement, a phenotype that has never been reported in CLN5 Batten disease. Lysosomal trafficking is key to maintain local degradation of cellular wastes, especially in long neuronal projections and our results from the human neuronal model present a key finding to understand the underlying lysosomal pathology in neurodegenerative diseases

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Patient-Derived Induced Pluripotent Stem Cell Models for Phenotypic Screening in the Neuronal Ceroid Lipofuscinoses

Molecules ◽

10.3390/molecules26206235 ◽

2021 ◽

Vol 26 (20) ◽

pp. 6235

Author(s):

Ahmed Morsy ◽

Angelica V. Carmona ◽

Paul C. Trippier

Keyword(s):

Neuronal Ceroid Lipofuscinosis ◽

Batten Disease ◽

Induced Pluripotent Stem Cell ◽

Epileptic Seizures ◽

Premature Death ◽

Model Systems ◽

Neuronal Ceroid Lipofuscinoses ◽

Phenotypic Screening ◽

Lysosomal Storage ◽

Induced Pluripotent

Batten disease or neuronal ceroid lipofuscinosis (NCL) is a group of rare, fatal, inherited neurodegenerative lysosomal storage disorders. Numerous genes (CLN1–CLN8, CLN10–CLN14) were identified in which mutations can lead to NCL; however, the underlying pathophysiology remains elusive. Despite this, the NCLs share some of the same features and symptoms but vary in respect to severity and onset of symptoms by age. Some common symptoms include the progressive loss of vision, mental and motor deterioration, epileptic seizures, premature death, and in the rare adult-onset, dementia. Currently, all forms of NCL are fatal, and no curative treatments are available. Induced pluripotent stem cells (iPSCs) can differentiate into any cell type of the human body. Cells reprogrammed from a patient have the advantage of acquiring disease pathogenesis along with recapitulation of disease-associated phenotypes. They serve as practical model systems to shed new light on disease mechanisms and provide a phenotypic screening platform to enable drug discovery. Herein, we provide an overview of available iPSC models for a number of different NCLs. More specifically, we highlight findings in these models that may spur target identification and drug development.

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A Human Induced Pluripotent Stem Cell-Derived Isogenic Model of Huntington’s Disease Based on Neuronal Cells Has Several Relevant Phenotypic Abnormalities

Journal of Personalized Medicine ◽

10.3390/jpm10040215 ◽

2020 ◽

Vol 10 (4) ◽

pp. 215

Author(s):

Tuyana Malankhanova ◽

Lyubov Suldina ◽

Elena Grigor’eva ◽

Sergey Medvedev ◽

Julia Minina ◽

...

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Neurodegenerative Disorder ◽

Microscopic Analysis ◽

Induced Pluripotent Stem Cell ◽

Cell System ◽

Neural Cells ◽

Electron Microscopic ◽

Healthy Human ◽

Induced Pluripotent

Huntington’s disease (HD) is a severe neurodegenerative disorder caused by a CAG triplet expansion in the first exon of the HTT gene. Here we report the introduction of an HD mutation into the genome of healthy human embryonic fibroblasts through CRISPR/Cas9-mediated homologous recombination. We verified the specificity of the created HTT-editing system and confirmed the absence of undesirable genomic modifications at off-target sites. We showed that both mutant and control isogenic induced pluripotent stem cells (iPSCs) derived by reprogramming of the fibroblast clones can be differentiated into striatal medium spiny neurons. We next demonstrated phenotypic abnormalities in the mutant iPSC-derived neural cells, including impaired neural rosette formation and increased sensitivity to growth factor withdrawal. Moreover, using electron microscopic analysis, we detected a series of ultrastructural defects in the mutant neurons, which did not contain huntingtin aggregates, suggesting that these defects appear early in HD development. Thus, our study describes creation of a new isogenic iPSC-based cell system that models HD and recapitulates HD-specific disturbances in the mutant cells, including some ultrastructural features implemented for the first time.

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Analysis of Gene Expression in Induced Pluripotent Stem Cell-Derived Human Neurons Exposed to Botulinum Neurotoxin A Subtype 1 and a Type A Atoxic Derivative

PLoS ONE ◽

10.1371/journal.pone.0111238 ◽

2014 ◽

Vol 9 (10) ◽

pp. e111238 ◽

Cited By ~ 3

Author(s):

Jacob M. Scherf ◽

Xiaoyang Serene Hu ◽

William H. Tepp ◽

Konstantin Ichtchenko ◽

Eric A. Johnson ◽

...

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Pluripotent Stem Cell ◽

Botulinum Neurotoxin ◽

Induced Pluripotent Stem Cell ◽

Type A ◽

Botulinum Neurotoxin A ◽

Human Neurons ◽

Induced Pluripotent

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From “Directed Differentiation” to “Neuronal Induction”: Modeling Neuropsychiatric Disease

Biomarker Insights ◽

10.4137/bmi.s20066 ◽

2015 ◽

Vol 10s1 ◽

pp. BMI.S20066 ◽

Cited By ~ 4

Author(s):

Seok-Man Ho ◽

Aaron Topol ◽

Kristen J. Brennand

Keyword(s):

Psychiatric Disorders ◽

Neurological Diseases ◽

Disease Onset ◽

Induced Pluripotent Stem Cell ◽

Directed Differentiation ◽

Neuropsychiatric Disease ◽

Somatic Cell Reprogramming ◽

Human Neurons ◽

Induced Pluripotent

Aberrant behavior and function of neurons are believed to be the primary causes of most neurological diseases and psychiatric disorders. Human postmortem samples have limited availability and, while they provide clues to the state of the brain after a prolonged illness, they offer limited insight into the factors contributing to disease onset. Conversely, animal models cannot recapitulate the polygenic origins of neuropsychiatric disease. Novel methods, such as somatic cell reprogramming, deliver nearly limitless numbers of pathogenic human neurons for the study of the mechanism of neuropsychiatric disease initiation and progression. First, this article reviews the advent of human induced pluripotent stem cell (hiPSC) technology and introduces two major methods, “directed differentiation” and “neuronal induction,” by which it is now possible to generate neurons for modeling neuropsychiatric disease. Second, it discusses the recent applications, and the limitations, of these technologies to in vitro studies of psychiatric disorders.

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Hyperexcitable phenotypes in iPSC-derived neurons from patients with 15q11-q13 duplication syndrome, a genetic form of autism

10.1101/286336 ◽

2018 ◽

Cited By ~ 4

Author(s):

James J. Fink ◽

Jeremy D. Schreiner ◽

Judy E. Bloom ◽

Dylan S. Baker ◽

Tiwanna M. Robinson ◽

...

Keyword(s):

Action Potential ◽

Induced Pluripotent Stem Cell ◽

Autism Spectrum ◽

Resting Membrane Potential ◽

Motor Deficits ◽

Action Potential Firing ◽

Potential Firing ◽

Synaptic Event ◽

Induced Pluripotent ◽

Duplication Syndrome

AbstractChromosome 15q11-q13 duplication syndrome (Dup15q) is a neurogenetic disorder caused by duplications of the maternal copy of this region. In addition to hypotonia, motor deficits, and language impairments, Dup15q patients commonly meet the criteria for autism spectrum disorder (ASD) and have a high prevalence of seizures. Here, we explored mechanisms of hyperexcitability in neurons derived from induced pluripotent stem cell (iPSC) lines from Dup15q patients. Maturation of resting membrane potential in Dup15q-derived neurons was similar to neurons from unaffected control subjects, but Dup15q neurons had delayed action potential maturation and increased synaptic event frequency and amplitude. Dup15q neurons also showed impairments in activity-dependent synaptic plasticity and homeostatic synaptic scaling. Finally, Dup15q neurons showed an increased frequency of spontaneous action potential firing compared to control neurons, in part due to disruption of KCNQ2 channels. Together these data point to multiple mechanisms underlying hyperexcitability that may provide new targets for the treatment of seizures and other phenotypes associated with Dup15q.

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Genome-wide molecular effects of the neuropsychiatric 16p11 CNVs in an iPSC-to-iN neuronal model

10.1101/2020.02.09.940965 ◽

2020 ◽

Author(s):

Thomas R. Ward ◽

Xianglong Zhang ◽

Louis C. Leung ◽

Bo Zhou ◽

Kristin Muench ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Cell Model ◽

Copy Number Variants ◽

Induced Pluripotent Stem Cell ◽

Neuronal Model ◽

Autism Spectrum ◽

Genome Wide ◽

Induced Pluripotent ◽

Methylation Patterns

AbstractCopy number variants (CNVs), either deletions or duplications, at the 16p11.2 locus in the human genome are known to increase the risk for autism spectrum disorders (ASD), schizophrenia, and for several other developmental conditions. Here, we investigate the global effects on gene expression and DNA methylation using a 16p11.2 CNV patient-derived induced pluripotent stem cell (iPSC) to induced neuron (iN) cell model system. This approach revealed genome-wide and cell-type specific alterations to both gene expression and DNA methylation patterns and also yielded specific leads on genes potentially contributing to some of the known 16p11.2 patient phenotypes. PCSK9 is identified as a possible contributing factor to the symptoms seen in carriers of the 16p11.2 CNVs. The protocadherin (PCDH) gene family is found to have altered DNA methylation patterns in the CNV patient samples. The iPSC lines used for this study are available through a repository as a resource for research into the molecular etiology of the clinical phenotypes of 16p11.2 CNVs and into that of neuropsychiatric and neurodevelopmental disorders in general.

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Sodium channel Nav1.2-L1342P variant displaying complex biophysical properties renders hyperexcitability of cortical neurons derived from human iPSCs

10.1101/2021.01.18.427192 ◽

2021 ◽

Author(s):

Zhefu Que ◽

Maria I. Olivero-Acosta ◽

Jingliang Zhang ◽

Muriel Eaton ◽

William C. Skarnes ◽

...

Keyword(s):

Sodium Channel ◽

Cortical Neurons ◽

Sodium Current ◽

Electrode Array ◽

Induced Pluripotent Stem Cell ◽

Epileptic Seizures ◽

Action Potential Firing ◽

Human Neurons ◽

Human Ipscs

AbstractWith the wide adoption of whole-exome sequencing in children having seizures, an increasing number of SCN2A variants has been revealed as possible genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene SCN2A, is strongly expressed in the pyramidal excitatory neurons and supports action potential firing. One recurrent SCN2A variant is L1342P, which was identified in multiple patients with early-onset encephalopathy and intractable seizures. Our biophysical analysis and computational modeling predicted gain-of-function features of this epilepsy-associated Nav1.2 variant. However, the mechanism underlying L1342P mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human induced pluripotent stem cell (hiPSC) line, in which L1342P was engineered by CRISPR/Cas9 mediated genome-editing. Using patch-clamping and micro-electrode array (MEA) recording, we found that the cortical neurons derived from hiPSCs carrying heterozygous L1342P variant presented significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, showing clear hyperexcitability phenotypes. Interestingly, the L1342P neuronal culture displayed a degree of resistance to the anti-seizure medication (phenytoin), which likely recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, was able to potently alleviate spontaneous and chemical-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures, and demonstrate the utility of genome-edited hiPSCs as an in vitro platform to advance personalized phenotyping and drug discovery.

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Two engineered AAV capsid variants for efficient transduction of human cortical neurons directly converted from iPSC

10.1101/2021.09.06.459070 ◽

2021 ◽

Author(s):

Sandra Fischer ◽

Jonas Weinmann ◽

Frank Gillardon

Keyword(s):

Cortical Neurons ◽

Transduction Efficiency ◽

Adeno Associated Virus ◽

Viral Genomes ◽

Glutamatergic Neurons ◽

Human Neurons ◽

Human Cell Cultures ◽

Primary Astrocytes ◽

Induced Pluripotent ◽

Viral Titers

Recombinant adeno-associated virus (AAV) is the most widely used vector for gene therapy in clinical trials. To increase transduction efficiency and specificity, novel engineered AAV variants with modified capsid sequences are evaluated in human cell cultures and non-human primates. In the present study, we tested two novel AAV capsid variants, AAV2-NNPTPSR and AAV9-NVVRSSS, in human cortical neurons, which were directly converted from human induced pluripotent stem cells and cocultured with rat primary astrocytes. AAV2-NNPTPSR variant efficiently transduced both induced human cortical glutamatergic neurons and induced human cortical GABAergic interneurons. By contrast, AAV9-NVVRSSS variant transduced both induced human cortical neurons and cocultured rat primary astrocytes. High viral titers (1x10E5 viral genomes per cell) caused a significant decrease in viability of induced human cortical neurons. Low viral titers (1x10E4 viral genomes per cell) lead to a significant increase in the neuronal activity marker c-Fos in transduced human neurons following treatment with a potassium channel blocker, which may indicate functional alterations induced by viral transduction and/or transgene expression.

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Autologous induced pluripotent stem cell-derived four-organ-chip

Future Science OA ◽

10.2144/fsoa-2019-0065 ◽

2019 ◽

Vol 5 (8) ◽

pp. FSO413 ◽

Cited By ~ 14

Author(s):

Anja Patricia Ramme ◽

Leopold Koenig ◽

Tobias Hasenberg ◽

Christine Schwenk ◽

Corinna Magauer ◽

...

Keyword(s):

Growth Factors ◽

Drug Testing ◽

Induced Pluripotent Stem Cell ◽

Neuronal Model ◽

Microphysiological Systems ◽

Disease Induction ◽

Induced Pluripotent ◽

Organ Models ◽

Model Platform ◽

Tissue Models

Microphysiological systems play a pivotal role in progressing toward a global paradigm shift in drug development. Here, we designed a four-organ-chip interconnecting miniaturized human intestine, liver, brain and kidney equivalents. All four organ models were predifferentiated from induced pluripotent stem cells from the same healthy donor and integrated into the microphysiological system. The coculture of the four autologous tissue models in one common medium deprived of tissue specific growth factors was successful over 14-days. Although there were no added growth factors present in the coculture medium, the intestine, liver and neuronal model maintained defined marker expression. Only the renal model was overgrown by coexisting cells and did not further differentiate. This model platform will pave the way for autologous coculture cross-talk assays, disease induction and subsequent drug testing.

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