Gene dosage-dependent rescue of HSP neurite defects in SPG4 patients' neurons

Abstract

The hereditary spastic paraplegias (HSPs) are a heterogeneous group of motorneuron diseases characterized by progressive spasticity and paresis of the lower limbs. Mutations in Spastic Gait 4 (SPG4), encoding spastin, are the most frequent cause of HSP. To understand how mutations in SPG4 affect human neurons, we generated human induced pluripotent stem cells (hiPSCs) from fibroblasts of two patients carrying a c.1684C>T nonsense mutation and from two controls. These SPG4 and control hiPSCs were able to differentiate into neurons and glia at comparable efficiency. All known spastin isoforms were reduced in SPG4 neuronal cells. The complexity of SPG4 neurites was decreased, which was paralleled by an imbalance of axonal transport with less retrograde movement. Prominent neurite swellings with disrupted microtubules were present in SPG4 neurons at an ultrastructural level. While some of these swellings contain acetylated and detyrosinated tubulin, these tubulin modifications were unchanged in total cell lysates of SPG4 neurons. Upregulation of another microtubule-severing protein, p60 katanin, may partially compensate for microtubuli dynamics in SPG4 neurons. Overexpression of the M1 or M87 spastin isoforms restored neurite length, branching, numbers of primary neurites and reduced swellings in SPG4 neuronal cells. We conclude that neurite complexity and maintenance in HSP patient-derived neurons are critically sensitive to spastin gene dosage. Our data show that elevation of single spastin isoform levels is sufficient to restore neurite complexity and reduce neurite swellings in patient cells. Furthermore, our human model offers an ideal platform for pharmacological screenings with the goal to restore physiological spastin levels in SPG4 patients.

GSK3ß‐dependent dysregulation of neurodevelopment in SPG11‐patient induced pluripotent stem cell model

“…We hypothesized that a loss‐of‐function mechanism of spatacsin is responsible for this proliferation deficit and confirmed this by knockdown of spatacsin in SH‐SY5Y cells. Whereas some HSP‐related genes, such as spastin (SPG4), spartin (SPG20), and spastizin (SPG15), are localized in the midbody and appear to facilitate cytokinesis during cell division,33, 34, 35 to our knowledge, proliferation of human cortical progenitors has only been studied in SPG4‐NPCs, where no proliferation deficit matching a pure spastic paraplegia phenotype was present 19. Our data point toward distinct temporal functions of spatacsin, causing axonal transport deficits in corticospinal projections in mature neurons in vitro, and most likely during adulthood, in patients 12.…”

“…Lysates were cleared by centrifugation at 11,000 g for 10 minutes at 4ºC, and immunoblot analysis was performed using the protocol described earlier 12, 19…”

Kdm6b and Pmepa1 as Targets of Bioelectrically and Behaviorally Induced Activin A Signaling

“…Human embryonic stem cells (HUES6, passages 24-28) were cultured under feeder-free conditions on Matrigel (BD Biosciences, Heidelberg, Germany) in mTeSR1 cell culture medium (Stemcell Technologies, Cologne, Germany) and passaged every 6-7 days by collagenase IV (200 U/ml) treatment (15 min), and a manual disintegration step using 1-ml glass pipettes. Neuronal differentiation was performed as previously described [24]. Neurons were cultured for 4 weeks, with half of the medium being replaced by fresh medium every week before they were treated with 50 ng/ml activin A.…”

“…To determine if the regulation is conserved in human neurons, human embryonic stem cells (HUES6) were differentiated into neural precursor cells (NPCs) using an embryoid bodybased protocol [24]. These cells stained positively for typical NPC markers like SOX2 and Nestin (Fig.…”

Re-engineered RNA-Guided FokI-Nucleases for Improved Genome Editing in Human Cells

“…HEK293T cells were chosen for their ease of handling, low maintenance costs, and because they have been frequently used in CRISPR/Cas9-related studies. 8,15,17 H9 human embryonic stem cells were cultured on growth-factorreduced Matrigel (Corning) in mTeSR1 (STEMCELL Technologies) at 37 C with 5% CO 2 as previously described 55,56 and passaged using ReLeSR (STEMCELL Technologies). For the RFX6 disruption assay, Y-27632 (10 mM; Tocris) pre-treated H9 cells were dispersed with Accutase to generate single-cell suspensions.…”