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Cerebrospinal Fluid from Patients with Sporadic Amyotrophic Lateral Sclerosis Induces Degeneration of Motor Neurons Derived from Human Embryonic Stem Cells

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Abstract

Disease modeling has become challenging in the context of amyotrophic lateral sclerosis (ALS), as obtaining viable spinal motor neurons from postmortem patient tissue is an unlikely possibility. Limitations in the animal models due to their phylogenetic distance from human species hamper the success of translating possible findings into therapeutic options. Accordingly, there is a need for developing humanized models as a lead towards identifying successful therapeutic possibilities. In this study, human embryonic stem cells—BJNHem20—were differentiated into motor neurons expressing HB9, Islet1, and choline acetyl transferase using retinoic acid and purmorphamine. These motor neurons discharged spontaneous action potentials with two different frequencies (< 5 and > 5 Hz), and majority of them were principal neurons firing with < 5 Hz. Exposure to cerebrospinal fluid from ALS patients for 48 h induced several degenerative changes in the motor neurons as follows: cytoplasmic changes such as beading of neurites and vacuolation; morphological alterations, viz., dilation and vacuolation of mitochondria, curled and closed Golgi architecture, dilated endoplasmic reticulum, and chromatin condensation in the nucleus; lowered activity of different mitochondrial complex enzymes; reduced expression of brain-derived neurotrophic factor; up-regulated neurofilament phosphorylation and hyperexcitability represented by increased number of spikes. All these changes along with the enhanced expression of pro-apoptotic proteins—Bax and caspase 9—culminated in the death of motor neurons.

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Acknowledgements

We are grateful to Prof. Inamdar, JNCASR, for providing the human embryonic stem cell line-BJNHem20. We are thankful to Ms. Jessena Ponmalar for her assistance in conducting mitochondrial assays and Dr. Mariamma Philip for the statistical analysis related to electrophysiology experiments.

Funding

This study was supported by National Institute of Mental Health and Neurosciences, Bengaluru, India. Senior Research Fellowship for SR was funded by Indian Council of Medical Research (ICMR) (File No. 81/03/2013/SCRT/BMS).

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Authors and Affiliations

  1. Department of Neurophysiology, National Institute of Mental Health and Neurosciences, Bengaluru, India

    Rajendrarao Sumitha, Rajesh K. Sabitha, Phalguni A. Alladi, Laxmi T. Rao, T. N. Sathyaprabha & Trichur R. Raju

  2. Department of Neurovirology, National Institute of Mental Health and Neurosciences, Bengaluru, India

    Venkataswamy M. Manjunatha

  3. Department of Neurology, National Institute of Mental Health and Neurosciences, Bengaluru, India

    A. Nalini

  4. Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bengaluru, India

    B. K. Chandrasekhar Sagar

  5. School of Mental Health and Neuroscience, Maastricht University Medical Center, Maastricht, The Netherlands

    Harry W. M. Steinbusch & Boris W. Kramer

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  1. Rajendrarao Sumitha
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  2. Venkataswamy M. Manjunatha
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  3. Rajesh K. Sabitha
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  4. Phalguni A. Alladi
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  5. A. Nalini
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  6. Laxmi T. Rao
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  7. B. K. Chandrasekhar Sagar
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  8. Harry W. M. Steinbusch
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  9. Boris W. Kramer
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  10. T. N. Sathyaprabha
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  11. Trichur R. Raju
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Corresponding author

Correspondence to Trichur R. Raju.

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This study was approved by the Institutional Ethics Committee and Steering Committee of Stem Cell Task Force, Department of Biotechnology, Government of India.

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The authors declare that they have no conflict of interest.

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Sumitha, R., Manjunatha, V.M., Sabitha, R.K. et al. Cerebrospinal Fluid from Patients with Sporadic Amyotrophic Lateral Sclerosis Induces Degeneration of Motor Neurons Derived from Human Embryonic Stem Cells. Mol Neurobiol 56, 1014–1034 (2019). https://doi.org/10.1007/s12035-018-1149-y

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  • DOI: https://doi.org/10.1007/s12035-018-1149-y

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