Multiparametric rapid screening of neuronal process pathology for drug target identification in HSP patient-specific neurons.

Axonal degeneration is a key pathology of neurodegenerative diseases, including hereditary spastic paraplegia (HSP), a disorder characterized by spasticity in the lower limbs. Treatments for HSP and other neurodegenerative diseases are mainly symptomatic. While iPSC-derived neurons are valuable for drug discovery and target identification, these applications require robust differentiation paradigms and rapid phenotypic read-outs ranging between hours and a few days. Using spastic paraplegia type 4 (SPG4, the most frequent HSP subtype) as an exemplar, we here present three rapid phenotypic assays for uncovering neuronal process pathologies in iPSC-derived glutamatergic cortical neurons. Specifically, these assays detected a 51% reduction in neurite outgrowth and a 60% increase in growth cone area already 24 hours after plating; axonal swellings, a hallmark of HSP pathology, was discernible after only 5 days. Remarkably, the identified phenotypes were neuron subtype-specific and not detectable in SPG4-derived GABAergic forebrain neurons. We transferred all three phenotypic assays to a 96-well setup, applied small molecules and found that a liver X receptor (LXR) agonist rescued all three phenotypes in HSP neurons, providing a potential drug target for HSP treatment. We expect this multiparametric and rapid phenotyping approach to accelerate development of therapeutic compounds for HSP and other neurodegenerative diseases.

Plastin 3 is upregulated in iPSC-derived motoneurons from asymptomatic SMN1-deleted individuals.

Spinal muscular atrophy (SMA) is a devastating motoneuron (MN) disorder caused by homozygous loss of SMN1. Rarely, SMN1-deleted individuals are fully asymptomatic despite carrying identical SMN2 copies as their SMA III-affected siblings suggesting protection by genetic modifiers other than SMN2. High plastin 3 (PLS3) expression has previously been found in lymphoblastoid cells but not in fibroblasts of asymptomatic compared to symptomatic siblings. To find out whether PLS3 is also upregulated in MNs of asymptomatic individuals and thus a convincing SMA protective modifier, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of three asymptomatic and three SMA III-affected siblings from two families and compared these to iPSCs from a SMA I patient and control individuals. MNs were differentiated from iPSC-derived small molecule neural precursor cells (smNPCs). All four genotype classes showed similar capacity to differentiate into MNs at day 8. However, SMA I-derived MN survival was significantly decreased while SMA III- and asymptomatic-derived MN survival was moderately reduced compared to controls at day 27. SMN expression levels and concomitant gem numbers broadly matched SMN2 copy number distribution; SMA I presented the lowest levels, whereas SMA III and asymptomatic showed similar levels. In contrast, PLS3 was significantly upregulated in mixed MN cultures from asymptomatic individuals pinpointing a tissue-specific regulation. Evidence for strong PLS3 accumulation in shaft and rim of growth cones in MN cultures from asymptomatic individuals implies an important role in neuromuscular synapse formation and maintenance. These findings provide strong evidence that PLS3 is a genuine SMA protective modifier.