Blocking Calpain Enzyme Found to Increase SMN Protein in Preclinical Models

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by Steve Bryson PhD |

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In cell- and mouse-based models of spinal muscular atrophy (SMA), reduced levels of the SMN protein — a hallmark of the disease — altered both the calpain enzyme system and autophagy, two major regulatory pathways of the cell, a study has discovered. 

Researchers found that blocking calpain increased the levels of SMN protein, suggesting this pathway may be a therapeutic target for SMA treatment. 

The study, “Calpain system is altered in survival motor neuron-reduced cells from in vitro and in vivo spinal muscular atrophy models,” was published in the journal Nature Cell Death & Disease

The most common types of SMA are caused by a mutation in the SMN1 gene, which provides instructions for the SMN protein. SMN protein plays an essential role in the maintenance of specialized nerve cells called motor neurons.

Motor neurons carry instructions from the brain to the spinal cord to control voluntary muscle movement, and a lack of SMN protein leads to the degeneration of the spinal cord (lower) motor neurons.

“Although the genetic causes of SMA are well-known, the mechanisms underlying lower [motor neuron] degeneration remain unclear,” the researchers wrote. 

Studies have suggested motor neuron degeneration is caused by alterations in autophagy — a process responsible for the degradation and recycling of damaged and unwanted proteins and other cell structures. 

In turn, there is evidence that autophagy is controlled by a group of calcium-dependent enzymes known as calpains. These enzymes modulate critical signaling pathways, and the overactivation of calpains has been implicated in neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. 

In this context, researchers at the University of Lleida in Spain recently reported that blocking calpain increased SMN protein levels in spinal motor neuron cells and improved the motor control and life span in two SMA mouse models. 

“Based on these results, the aim of the present work was to investigate the calpain pathway in SMN-reduced [motor neurons],” the researchers wrote.

The first step was to find out if the calpain pathway is altered in spinal cord motor neurons with reduced SMN protein isolated from both SMA mouse models. 

In one mouse model, the entire SMN1 gene was deleted while the other, a mouse model called SMNdelta7, had a portion known as exon 7 of the SMN1 gene deleted, which is also deleted in about 95% of people with SMA

In spinal cord motor neurons isolated from both these mice, as expected, the level of SMN protein was significantly reduced compared to neurons from healthy mice controls. 

While the levels of calpain protein were not increased, the activity of calpain was significantly increased compared to controls. Furthermore, the amount of a natural calpain inhibitor that modulates calpain activity, called calpastatin, was not affected, pointing to an unknown calpain activation mechanism. 

To further investigate changes to the calpain pathway, two human SMA cell-based models were used; fibroblasts, the most common cells of connective tissue, and motor neurons generated from stem cells. Both cell lines were initially isolated from SMA patients and had reduced levels of SMN protein.

In SMA fibroblasts, the activity of calpain was reduced. In contrast, in SMA stem-cell-derived motor neurons, calpain activity was significantly higher, while at the same time, the overall calpain protein levels were lower. 

To investigate changes in vivo, the SMNdelta7 mice were subcutaneously (under-the-skin) injected with a chemical called calpeptin which blocks calpain activity, starting at birth and for eight days after. Spinal cord tissue was then isolated and analyzed. 

In untreated mice, the calpain activity significantly increased while, in contrast, the calpain activity in mice treated with calpeptin was the same as control (healthy) mice. Importantly, the level of SMN protein was higher in spinal cord motor neurons of calpeptin-treated mice. 

“Calpains are considered as modulator … that can regulate protein functions, and therefore various cellular pathways, including autophagy,” the researchers wrote.

“In many disease environments and models, calpains are known to negatively regulate autophagy. Consequently, enhanced calpain activation can contribute to the compromised activation of this degradation pathway,” they added.

As such, to investigate alterations to autophagy, the level of a protein central to the autophagy pathway, called LC3, was measured.

In motor neurons isolated from SMNdelta7 mice, LC3 levels were significantly increased compared with those from control mice. 

Incubating these neurons with the calpain blocker calpeptin led to an increase in LC3 in the body of the neurons called the soma, while reducing LC3 in neurites — projections leading away from the neuron cell body. 

According to the authors, “[t]his effect would prevent the neurite collapse that can lead to [motor neuron] degeneration.” 

“The present study found an increase of calpain activity in SMN-reduced cells,” the researchers wrote. 

“We suggest that the positive modifier effect of in vivo calpeptin treatment on SMA mice survival and motor phenotype could be the result of SMN protein and LC3 [autophagy] protein regulation in SMA [motor neurons] and could offer a viable therapeutic approach,” they said. 

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