Modelling a complex condition such as ALS in a laboratory is an understandably difficult concept, and it is perhaps unsurprising that we don’t yet have the perfect model. This session early on a Saturday morning looked at the different approaches to using cells in the lab to investigate ALS.
The first speaker and one of the chairs of this session, Dr Kevin Eggan from Harvard, started us off by describing how iPSCs (induced pluripotent stem cells) can be a useful tool in the study of ALS. The technique for creating and using iPSCs was excellently reviewed by Dr Eggan in Neuron (2011; 70(4):626-44). Patient cells exposed to different growth factors can be reverted to a pluripotent state and then turned into motor neurons, with the added benefit of retaining any genetic markers from the patient.
Dr Eggan’s lab looked at iPSC-derived motor neurons from two patients with SOD1 mutations. These patient motor neurons appeared to be more electrically active than motor neurons from healthy controls, and if the SOD1 mutation was corrected in the patient motor neurons (using zinc finger nucleases) then their cells behaved similarly to the control patients’ cells. Increased electrical activity in motor neurons with a SOD1 mutation might be due to decreased potassium currents. A drug currently used for epilepsy, retigabine, acts on potassium channels. Treatment of patient motor neurons with retigabine opened the potassium channels and returned the SOD1 trace to similar to controls. Using iPSCs from patients with SOD1 mutations has led to this new possible treatment, retigabine, going forward into clinical trials. Dr Eggan and his lab are hopeful that looking at iPSCs from ALS patients with other genetic backgrounds could hold similar clues for future treatment.
A further example of how iPSCs can be used in ALS research came from Dr Mutihac, based in Oxford. Using the same method of reprogramming patient skin fibroblasts and differentiating into motor neurons, Dr Mutihac is looking at the behaviour of these motor neurons, and calcium signalling in particular. Skin cells were taken from patients with the C9ORF72 expansion, which was not lost in the iPSC-differentiation process. Around 30-40% of the patient motor neurons developed intracellular RNA foci, a marker for ALS. There appeared to be dysregulation of calcium in the patient cells, with increased calcium currents in patient’s motor neurons (but not in similarly derived cortical neurons) and formation of stress granules. Dr Mutihac also reported a small but significant increase in TDP-43 mislocalisation in patient motor neurons compared to controls. This work is ongoing, but Dr Mutihac proposes that these iPSCs will be a valuable tool for drug screening in the future.
Although iPSC models are useful in ALS research, they take many months to develop and so other cellular models are often used instead. The next speaker was Dr Makkar from the Barrow Neurological Institute in the USA, who is investigating the role of RBM45 in ALS, a protein which has been found in increased levels in the cerebrospinal fluid of ALS patients and also within TDP-43 and ubiquitin-positive aggregates. Using primary rat motor neurons and human neuroblastoma cell lines, this group has shown that nuclear protein RBM45 forms granules in the cytoplasm in response to oxidative stress. Oxidative stress is known to be involved in ALS pathophysiology, and Dr Makkar showed that RBM45 is associated with the KEAP1/NRF2/ARE pathway (an intracellular pathway that reacts to oxidative stress leading to cell death) which has previously been implicated in ALS. Under stress, RBM45 was shown to bind to KEAP1 and is suggested to lead to increased expression of the KEAP1 protein leading to increased cell death. Dr Makkar intends to continue investigating why RBM45 is mislocalised from the nucleus and the nature of the cytoplasmic granules it forms.
In the final talk of this session, Dr Farg discussed how the hexanucleotide repeat expansions cause nucleolar stress in neuronal cell lines. Using GGGGCC repeats linked to a fluorescent protein, Dr Farg showed that longer repeats lead to increased aggregate formation and an increase in markers for nucleolar stress, such as depleted B23. B23 expression contributes to the DNA damage response, activating the PI3K/Akt pathway leading to DNA repair. Depleted B23 in response to GGGGCC repeat expression will therefore reduce the cell’s ability to repair its DNA. This furthers our understanding of how the GGGGCC repeat in C9ORF72 is linked to ALS.
This session showcased how important in vitro modelling is for basic research into ALS. Whether through human neuroblastoma cell lines, primary rat motor neurons or patient-iPSC-derived motor neurons, each of these talks showed how much information can be gathered from simple cell-based models, and how that is furthering our understanding of this disease.