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

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.

Cerebrospinal fluid cytotoxicity in amyotrophic lateral sclerosis: a systematic review of in vitro studies

“…Seven of these studies were quantitative in nature and observed significant increases in the levels of different apoptotic markers, including caspase-3 and caspase-9, following exposure to ALS–CSF compared to controls. However, the increase in Bcl-2 levels following ALS–CSF exposure did not reach statistical significance ( Ding et al , 2015 ; Sumitha et al , 2019 ). The remaining study, which only qualitatively evaluated apoptosis, reported the presence of TUNEL-stained nuclei in cells exposed to ALS–CSF, but not in those from control groups ( Vijayalakshmi et al , 2011 ).…”

“…In vitro culture models included spinal cord cultures, cortical neuron cultures and motor neuron cultures, as well as cell lines, such as NSC-34 (mouse spinal cord-neuroblastoma hybrid cell line), VSC 4.1 (cholinergic cAMP-differentiated motor neuron-neuroblastoma hybrid cell line) and U251 (human glioma cell line). One recent study also assessed cytotoxicity in motor neurons differentiated from human embryonic stem cells ( Sumitha et al , 2019 ).…”

“…Amongst the many lines of enquiry aiming to address this question, several studies have suggested that a neurotoxic cerebrospinal fluid (CSF) profile could be implicated in the disease process ( Shaw, 2002 ; Matias-Guiu et al , 2010 ; Sumitha et al , 2019 ; Tokuda et al , 2019 ). CSF from ALS patients has in fact been shown to exert cytotoxicity in vitro ( Tikka et al , 2002 ; Vijayalakshmi et al , 2009 ; Barber et al , 2011 ; Sumitha et al , 2019 ), and to provoke wide-ranging pathology, from neurofilament phosphorylation to musculoskeletal changes, when administered in vivo ( Shahani et al , 2004 ; Gomez-Pinedo et al , 2018 ; Shanmukha et al , 2018 ). While the cause of these findings remains unclear, they nevertheless suggest the presence of one or more potentially toxic factors in ALS–CSF, with possible involvement in disease spread ( Smith et al , 2015 ).…”

Neuro‐Cells therapy improves motor outcomes and suppresses inflammation during experimental syndrome of amyotrophic lateral sclerosis in mice

“…The upregulation of GSK‐3ß cascade is known to be functionally associated with pro‐inflammatory changes and microglial activation, which is a well‐established marker of ALS histopathology, and the FUS‐ALS forms in particular . GSK‐3ß activities are inversely related to neurotrophin expression, and its downregulation is another recognized molecular feature of human ALS . GSK‐3ß is also implicated in the mechanisms of microglial activation during neurodegeneration .…”

Defining novel functions for cerebrospinal fluid in ALS pathophysiology

“…Although CSF from ALS patients is now known to be constitutionally abnormal [ 18 , 27 ], with raised levels of proteins, including TDP-43 and neurofilaments [ 115 , 210 ], its toxicity started being recognised early on, when it was shown to significantly reduce the survival of rat primary neuronal cultures [ 45 ]. Since then, numerous studies have been performed on various cell types, including NSC-34 cell lines, and, more recently, hESC-derived and iPSC-derived motor neurons, showing greater degeneration when the cells were exposed to CSF from ALS patients than to CSF from control patients [ 33 , 136 , 189 , 197 ]. Interestingly, ALS-CSF was also shown in NSC-34 cells to result in TDP-43 mislocalisation to the cytoplasm, a feature that could be reversed by VEGF [ 174 ].…”