Unravelling the cellular mechanism of spinal muscular atrophy: from gene and modifiers to therapy

Spinal muscular atrophy (SMA) is a common neuromuscular disease characterised by weakness and wasting of muscles. People with the most severe form of the disease are unable to sit or walk and die within the first two years of life. Although the genetics of SMA are well understood, the cellular mechanisms involved are unclear and there is currently no cure. Professor Dr Brunhilde Wirth at the University of Cologne is using advanced genetics to discover SMA protective modifiers and various animal models to unravel the cellular mechanisms responsible for SMA and networks of protection, with the ultimate goal of developing novel therapeutics.

There are currently ~50,000 people living with SMA in Europe and the USA and, in the European population, one in every 35 people is a carrier of the disease. SMA is characterised by the progressive loss of motor neurons – specialised nerve cells that innervate muscle and stimulate contraction – and impaired neuromuscular junctions – the synapses between motor neurons and muscle cells. In patients with SMA, the muscles are under stimulated and so they weaken and waste away in a process called atrophy.

Survival motor neuron
People with SMA have a deficit of a vital protein called “survival motor neuron” (SMN). SMN is produced by all body cells and is important for their survival. However, motor neurons require fifty times more SMN than any other cell type. Most individuals with SMA have inherited two absent copies of the SMN1 gene (the gene responsible for SMN production) from their parents.

Humans have an almost identical second copy of the SMN gene, called SMN2. This gene also produces SMN protein but, due to a problem with the splicing process, only 10% of the protein is functional. In SMA-affected individuals, SMN2 is the only source of SMN, so its copy number (humans can have between one and six copies) affects the severity of disease. For example, patients with two absent SMN1 genes and only two copies of SMN2 will usually have the most severe form of SMA (type I). These babies will develop symptoms before six months of age, will never be able to sit or walk, will be dependent on respiratory and nutritional support, and have a life expectancy of usually less than two years. People with two absent SMN1 genes but three copies of SMN2 usually have an intermediate SMA and learn to sit but never to walk. Those with two absent SMN1 genes and four to six copies of SMN2 usually have a mild form of the disease (type III or IV). Onset of symptoms is not until later in childhood or adulthood and patients are able to sit and walk despite some muscle weakness.

Dr Wirth’s research highlights the power of protective modifiers to unveil the cellular mechanisms and develop novel
therapies for SMA

Phynotypically discordant families teach us a lot
In rare families, relatives of SMA-affected individuals may have no SMA symptoms despite carrying two absent SMN1 genes together with three or four SMN2 copies, a combination that would usually cause SMA. This suggests there are other factors involved in determining the severity of SMA.

By using advanced technologies to study the genes and proteins of family members in these SMA-discordant families, Professor Wirth has identified two proteins that act as SMA protective modifiers: Plastin 3 (PLS3) and Neurocalcin Delta (NCALD).

Family members with two absent SMN1 genes who are unaffected by SMA produce much more PLS3 than their affected relatives. Conversely, unaffected family members with two absent SMN1 genes produce much less NCALD than their affected counterparts. Both high PLS3 and low NCALD levels have been shown to protect against disease symptoms. For example, in one SMA-discordant family, in which two absent SMN1 genes and only four copies of SMN2 usually resulted in type III disease, low NCALD levels protected five family members across four generations.

The protective effects of high PLS3 and low NCALD have been corroborated by Prof Wirth’s group using animal models of SMA, including mice, worms and zebrafish. Here, high levels of PLS3 or low levels of NCALD were shown to reduce disease symptoms. Using these same animal models, the group also showed that both PLS3 and NCALD protect against SMA by restoring endocytosis – a process that is essential for recycling the synaptic vesicles involved in transmission of nerve impulses across the neuromuscular junctions, which is impaired in SMA.

People with the severe form of SMA need a combinatorial therapy and a systemic increase of SMN in every single cell

From gene to therapy
Having identified PLS3 and NCALD as protective modifiers, Prof Wirth’s group began to explore whether regulating production of these proteins could be used as a therapeutic strategy to treat SMA. The group used mouse models with either high PLS3 or low NCALD in combination with a low dose of SMN antisense oligonucleotides (ASOs), small molecules that increase SMN levels by targeting its production by the SMN2 gene. Both approaches dramatically reduced SMA symptoms. Strikingly, small amounts of SMN-ASOs in combination with low PLS3 increased animal survival from 14 days to over 250 days. This situation is comparable to severely affected type I SMA patients, where ASO therapy increases SMN production but not enough to cure the disease. In this instance, a combined therapy with a second agent, such as PLS3 or NCALD, could constitute a long term therapeutic option.

The first SMN ASO-based therapy (Spinraza) was recently FDA- and EMA-approved. Since the Wirth group have shown that a low dose of SMN-ASOs in combination with high PLS3 or low NCALD protects against even the most severe type of SMA in mice, regulation of these proteins could be used in combinatorial therapies with Spinraza to increase treatment efficacy.

Prof Wirth’s research highlights the power of protective modifiers to unveil the cellular mechanisms and develop novel therapies for SMA. The group are developing and using a number of different methods and technologies to identify the genetic cause of unsolved motor neuron disorders and to understand the genetic, biochemical, cellular and pathological basis of these disorders. They are also generating and using mouse models, zebrafish and, most recently, Drosophila (fruit fly) models as well as induced pluripotent stem cells to unveil the disease pathomechanism. Prof Wirth is now extending her research to search for the molecular cause of osteoporosis.