Sensory Neurons Do Not Induce Motor Neuron Loss in a Human Stem Cell Model of Spinal Muscular Atrophy

Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive disorder leading to paralysis and early death due to reduced SMN protein. It is unclear why there is such a profound motor neuron loss, but recent evidence from fly and mouse studies indicate that cells comprising the whole sensory-motor circuit may contribute to motor neuron dysfunction and loss. Here, we used induced pluripotent stem cells derived from SMA patients to test whether sensory neurons directly contribute to motor neuron loss. We generated sensory neurons from SMA induced pluripotent stem cells and found no difference in neuron generation or survival, although there was a reduced calcium response to depolarizing stimuli. Using co-culture of SMA induced pluripotent stem cell derived sensory neurons with control induced pluripotent stem cell derived motor neurons, we found no significant reduction in motor neuron number or glutamate transporter boutons on motor neuron cell bodies or neurites. We conclude that SMA sensory neurons do not overtly contribute to motor neuron loss in this human stem cell system.

Neurite Aggregation and Calcium Dysfunction in iPSC-Derived Sensory Neurons with Parkinson’s Disease-Related LRRK2 G2019S Mutation

“…Neurite aggregates have been observed in many different neurodegenerative diseases including PD ( MacLeod et al., 2006 ) and may be an indicator of early stages of axonopathy. Neurite aggregates may be a prominent feature of the LRRK2 G2019S mutation as the phenotype was not observed in SNCA (3×) iPSC-derived sensory neurons ( Figure 2 B) and was also absent from peripheral sensory neurons derived from other diseased iPSCs ( Schwab and Ebert, 2014 ). Immunocytochemistry for ATF3, a transcription factor activated in sensory neurons following nerve injury ( Tsujino et al., 2000 , Lindå et al., 2011 ), was not increased in LRRK2 G2019S cultures (data not shown), indicating minimal peripheral nerve damage.…”

“…iPSC-derived sensory neuron cultures were functionally tested using ratiometric live-cell calcium imaging using dual-wavelength fluorescent calcium indicator FURA-2AM (Life Technologies) to detect intracellular calcium levels as described previously ( Schwab and Ebert, 2014 ). Metafluor imaging software was used to detect and analyze intracellular calcium changes throughout the experiment (Molecular Devices), where a ≥20% increase in intracellular calcium from baseline constituted a response.…”

Is spinal muscular atrophy a disease of the motor neurons only: pathogenesis and therapeutic implications?

“…In contrast, other studies point to motor neuron dysfunction triggering secondary sensory and interneuron deficits in SMA [9,27,49,50]. In a cellular model employing iPSC-derived neurons, Schwab and Ebert (2014) (Table 1) have used a direct co-culture approach to assess whether sensory neurons established from SMA-derived iPS cell lines can lead to WT motor neuron loss [51]. They found that SMA iPSC-derived sensory neurons have an negligible impact on motor neuron survival, or expression of glutamate transporters (VGLUT1, VGLUT2) in neurites and cell bodies of motor neuron; concluding that SMN-deficient sensory neurons are not the primary trigger of motor neuron loss [51].…”

“…In a cellular model employing iPSC-derived neurons, Schwab and Ebert (2014) (Table 1) have used a direct co-culture approach to assess whether sensory neurons established from SMA-derived iPS cell lines can lead to WT motor neuron loss [51]. They found that SMA iPSC-derived sensory neurons have an negligible impact on motor neuron survival, or expression of glutamate transporters (VGLUT1, VGLUT2) in neurites and cell bodies of motor neuron; concluding that SMN-deficient sensory neurons are not the primary trigger of motor neuron loss [51]. As described above, it has been shown that motor neuron loss is preceded by a small reduction in VGLUT1+ bouton numbers on sensory neurons [24].…”

Model Systems of Motor Neuron Diseases As a Platform for Studying Pathogenic Mechanisms and Searching for Therapeutic Agents

“…iPSC lines with the SMA genotype were differentiated into sensory neurons. In this case, a decrease in the calcium response to depolarizing stimuli was observed, but the survival of these cells did not differ from that of the control group cells [ 125 ]. The co-culture of sensory neurons from SMA patients and motor neurons from healthy donors revealed no significant reduction in the number of motor neurons, as well as the formation of clusters of glutamate transport vesicles near the bodies of the motor neurons and neurites.…”