ALS Patient Stem Cells for Unveiling Disease Signatures of Motoneuron Susceptibility: Perspectives on the Deadly Mitochondria, ER Stress and Calcium Triad

Abstract

Amyotrophic lateral sclerosis (ALS) is a largely sporadic progressive neurodegenerative disease affecting upper and lower motoneurons (MNs) whose specific etiology is incompletely understood. Mutations in superoxide dismutase-1 (SOD1), TAR DNA-binding protein 43 (TARDBP/TDP-43) and C9orf72, have been identified in subsets of familial and sporadic patients. Key associated molecular and neuropathological features include ubiquitinated TDP-43 inclusions, stress granules, aggregated dipeptide proteins from mutant C9orf72 transcripts, altered mitochondrial ultrastructure, dysregulated calcium homeostasis, oxidative and endoplasmic reticulum (ER) stress, and an unfolded protein response (UPR). Such impairments have been documented in ALS animal models; however, whether these mechanisms are initiating factors or later consequential events leading to MN vulnerability in ALS patients is debatable. Human induced pluripotent stem cells (iPSCs) are a valuable tool that could resolve this "chicken or egg" causality dilemma. Relevant systems for probing pathophysiologically affected cells from large numbers of ALS patients and discovering phenotypic disease signatures of early MN susceptibility are described. Performing unbiased 'OMICS and high-throughput screening in relevant neural cells from a cohort of ALS patient iPSCs, and rescuing mitochondrial and ER stress impairments, can identify targeted therapeutics for increasing MN longevity in ALS.

Keywords: ALS; C9orf72; TDP43; calcium homeostasis; endoplasmic reticulum; iPSCs; mitochondria; motoneurons.

Figure 1

Oxidative stress, protein misfolding and mitochondrial dysfunction are closely related. Excessive production of reactive oxygen or nitrogen species (ROS/RNS), transcriptional dysregulation, protein misfolding and ER stress can arise as consequences of OS and mitochondrial stress. In addition these factors work in a feedback-loop further exacerbating mitochondrial stress and dysfunction. A significant amount of mitochondrial proteins, including those of the ETC, contain highly oxidizable iron-sulfur-clusters that upon exposure to OS can be severely affected in their folding and function. But, OS also triggers stress responses in other organelles, such as the ER and persistent stress and highly oxidative conditions impair the function and integrity of protein folding in the ER. As a result the formation of misfolded proteins is favored leading to an accumulation of insoluble cytosolic and mitochondrial aggregates, impaired interference with activity of the PDI and impaired axonal transport. During the course of these alterations the levels of ATP and intra-cellular calcium are affected. This change interferes with the Ca²⁺ and ATP sensitive mitochondrial fusion/fission machinery and microtubule based mechanisms of mitochondrial transport. Mitochondria accumulate in the cell soma in a fragmented and dysfunctional state leading to a dramatic reduction of mitochondria transported anterograde to the axon terminal. Given the size of motor neurons with long axonal extensions, the impaired axonal transport leads to a depletion of functional mitochondria at the axon terminal. With the axonal periphery no longer supplied with sufficient ATP distal synapses degenerate eventually and the MN dies. As a consequence myofibers no longer receive input from their corresponding MN and are prone to atrophy, manifesting in increased muscle weakness of ALS patients.

Figure 2

Protein misfolding triggers ER stress in neurons. Misfolded proteins trigger the unfolded protein response (UPR) of the ER, leading to cellular efforts in either refolding proteins to a normal state or ubiquitinating or degrading unrepairable proteins. Some proteins, i.e., TDP-43 or FUS escape degradation via proteasomes and autophagy resulting in large aggregates. Also, mutant TDP-43 and FUS accumulate in the cytoplasm. Continuous activation of UPR and other ER stress responses lead to ER dysfunction, fragmentation of the Golgi apparatus and protein trafficking defects. Adding to this, ROS and RNS released by ER and mitochondria, as well as excitotoxicity via glutamatergic synapses yield mitochondrial stress and fragmentation and further contribute to ER stress. As a result further formation of misfolded proteins is favored leading to an accumulation of insoluble cytosolic and mitochondrial aggregates, and an impaired activity of the PDI of the ER. Collectively, these events culminate with further overall neuronal dysfunction including dynein based transport inhibition. Ultimately, this results in a breakdown of energy supply for the distal axon terminal. Astroglial dysfunction or toxicity can contribute to MN degeneration as well, whether as initiating event or contributing factor remains to be determined. Activated astrocytes and microglia can trigger inflammatory responses resulting in further MN stress. Finally, a prion-like hypothesis posits that misfolded protein aggregates can spread from surrounding cells, either affected dying neurons or astrocytes, and infects MNs thereby initiating stress responses and ultimately apoptosis in the infected cells.

Figure 3

Possible causes of ALS. The most prevalent underlying reason for MN defects are genetic perturbagens, inherited or de novo mutations. Yet, the majority of ALS cases have not been linked to any mutation, suggesting that other effectors such as the environment may play into this as well. Thus MN phenotypes observed in ALS may arise from genetic and/or environmental perturbagens, as depicted in the top panel. Developmental anomalies may affect the structural integrity of neuronal cytoarchitecture as conferred by structural proteins, transport proteins, transmembrane proteins or by a disruption of RNA processing. Such defects can interfere or even prevent the formation of synapses between neurons or at the NMJ either directly within MN or indirectly by affecting neighboring glial cells. Triggering of autoimmune responses and chronic low-level inflammation may lead to MN degeneration as well and many of those developmental defects manifest in electrophysiological deficiencies such as a progressive decrease in voltage-activated Na⁺ and K⁺ currents correlated with a loss of functional outputs. Mitochondrial susceptibility due to ROS-induced OS in turn yields an inert vulnerability of MNs to excitotoxicity. An increased amount of mitochondrial stress in turn leads to mitochondrial fragmentation and ultimately cell death. Due to their large size and long neurite outgrowths, MNs are particularly sensitive to ion fluctuations, whether due to selective permeability for Ca²⁺ influx or lack of messenger clearance from the synaptic cleft. The dysregulation of intracellular Ca²⁺ levels has severe implications for MN function as well. Both, the ER and mitochondria function as buffers of cellular Ca²⁺ homeostasis. When intracellular Ca²⁺ levels increase, either by influx from the extracellular space or the ER and mitochondria, this triggers OS responses, their fragmentation and eventually progression of cell death signals that ultimately lead to loss of electrophysiological outputs. The generation of misfolded proteins and formation of aggregates, likewise affects the functional integrity of both organelles. Initially, misfolded proteins trigger the UPR in the ER to compensate for decreased protein translation and processing efficiency. Persistence of misfolded proteins that escape corrective degradation mechanisms cause accumulation in the cell and ultimately lead to the formation of insoluble aggregates interfering with cellular functions such as molecular transport and trafficking.

Figure 4

Molecular disease signatures of MNDs. ALS Patient derived cells, i.e., skin cells or whole blood allow for the generation of iPS cells and the subsequent differentiation into MNs via neural progenitors. Not only does iPS cell technology now allow for the large scale production of MNs but, conceptually, also the generation of all the diverse subtypes of upper and lower MNs affected in ALS. As a disease-in-a-dish model, patient-iPSC-derived motor neuron subtypes can aid in unraveling the molecular mechanisms involving mitochondrial and ER dysfunction, as a cause of ALS vs. being a secondary effect after disease onset. Systems biology approaches utilizing large scale genomics, transcriptomics and proteomics will help identify novel targets of MNDs such as ALS and SMA compared to iPSC derived MNs from control patients. For further analysis and validation of prominent gene targets, gene editing techniques creating reporter and genetic loss-of-function cell lines can facilitate the development of disease signatures of ALS patient iPSC-derived MNs. Particularly, collaborative efforts such as i.e., the NeuroLINCS initiative will enable the acquisition of large data sets of cellular phenotypes and drug screening assays. Building on established data from animal models this approach may dramatically accelerate the discovery of valid new therapeutic approaches and drug targets.