ALS Researchers Probe Cerebrospinal Fluid’s Role in Motor Neurons

The clear, colorless liquid that fills and surrounds the brain and spinal cord in people with amyotrophic lateral sclerosis (ALS) seems to induce early neurodegeneration events in healthy motor neurons, a study has found.

When motor neurons were continuously cultured with cerebrospinal fluid  from ALS patients, cells showed evidence of Golgi fragmentation — when their Golgi apparatus, an organelle that helps package proteins and other molecules to be exported from the cell, becomes fragmented and dispersed.

The study, “Human Spinal Motor Neurons Are Particularly Vulnerable to Cerebrospinal Fluid of Amyotrophic Lateral Sclerosis Patients,” was published in the International Journal of Molecular Sciences.

The cerebrospinal fluid (CSF) of ALS patients has been suspected of carrying factors that can cause disease, perhaps being a route for disseminating ALS to other motor neurons. Studies involving cells and animal models have demonstrated that CSF from patients may induce various disease aspects, including protein clumps, cell stress, abnormalities in the mitochondria and energy production, as well as Golgi fragmentation.

However, none of these studies was conducted in motor neurons derived from patients’ induced pluripotent stem cells (iPSCs), which now is considered the gold standard cell line for modeling ALS.

Because motor neurons cannot come directly from patients, iPSCs, which can be generated from patient’s blood or skin cells, are seen as a helpful alternative. As their name suggests, these cells are pluripotent — meaning that, like other stem cells, they are able to differentiate into other cell types.

As such, researchers can collect blood or skin cells from a person with ALS and use them to generate iPSCs, which can be differentiated into motor neurons. Since these motor neurons are derived from the iPSCs of an ALS patient, they will have the same genetic abnormalities, making them an apt model for study.

Researchers in Dresden, Germany, set out to explore the effects of CSF in this cell model of sporadic ALS. They used iPSC-derived motor neurons from three patients — one with FUS mutations, one with SOD1 mutations, and a third with TDP-43 mutations — as well as similarly generated motor neurons from healthy controls.

CSF samples were collected from 11 ALS patients and eight controls, who underwent CSF puncture due to non-inflammatory, non-neurodegenerative neurological disorders (e.g., headache). Apart from patients being older than controls (median 63 years vs. 47 years), the groups were generally similar, even in their CSF molecular composition.

The team noted that CSF from both patients and controls caused a significant proliferation of the neural progenitor cells that remained in culture after iPSCs had been induced to become motor neurons. The larger the amounts of CSF and the longer the duration of exposure to CSF, the greater the effect on these progenitor cells.

To avoid that, cells were left to differentiate into motor neurons for another 10 days, and only then were the experiments with CSF started. Because cells were cultured for long periods (six days), which required large amounts of CSF, the team used pooled CSF samples from ALS patients and from controls.

“Thus, we cannot answer the question as to whether single ALS patient-derived CSF might vary depending on clinical parameters such as ALS subtype, disease stage or even family background,” the researchers noted.

Overall, CSF from both patients and controls significantly reduced the proportion of neurons in culture — a 45% reduction for both ALS-CSF and control-CSF versus 69% for non-CSF treated cells — when motor neurons from healthy donors were used. A similar effect, as well as a lower proportion of motor neurons, was observed in motor neuron cultures from patients.

CSF treatment did not influence the neuronal networks established by motor neurons, or cause the formation of protein clumps, which is a hallmark of neurodegenerative diseases like ALS.

However, CSF from ALS patients induced significant Golgi fragmentation, an early event in the neurodegenerative process, in control motor neurons — but not in other control nerve cells. This effect was not observed on ALS motor neurons likely because the initial Golgi fragmentation before CSF treatment was already high.

“By using iPSC-derived MNs [motor neurons] from healthy controls and monogenetic forms of ALS we demonstrate a harmful effect of CSF from patients suffering from sporadic ALS on healthy donor-derived human MNs,” the researchers wrote.

“Golgi fragmentation — a typical finding in lower organism ALS models and human post mortem tissue of ALS patients — was induced solely by the addition of ALS- but not control-CSF. Strikingly, these changes occurred predominantly in MNs, the cell type primarily affected in ALS,” they concluded.