HDAC6 inhibition reverses axonal transport defects in motor neurons derived from FUS-ALS patients

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder due to selective loss of motor neurons (MNs). Mutations in the fused in sarcoma (FUS) gene can cause both juvenile and late onset ALS. We generated and characterized induced pluripotent stem cells (iPSCs) from ALS patients with different FUS mutations, as well as from healthy controls. Patient-derived MNs show typical cytoplasmic FUS pathology, hypoexcitability, as well as progressive axonal transport defects. Axonal transport defects are rescued by CRISPR/Cas9-mediated genetic correction of the FUS mutation in patient-derived iPSCs. Moreover, these defects are reproduced by expressing mutant FUS in human embryonic stem cells (hESCs), whereas knockdown of endogenous FUS has no effect, confirming that these pathological changes are mutant FUS dependent. Pharmacological inhibition as well as genetic silencing of histone deacetylase 6 (HDAC6) increase α-tubulin acetylation, endoplasmic reticulum (ER)–mitochondrial overlay, and restore the axonal transport defects in patient-derived MNs.

(C) Staining with different MN markers (Isl1, Chat, SMI-32, Synapsin1) at day 38 of cells differentiated from the isogenic control iPSC line and quantification of ISL1-positive cells, SMI-32-positive cells and CHAT-positive cells expressed relative to the total number of DAPI labeled cells. N=10 images per line. Data are represented as mean ± SEM. Scale bar = 20µm.

Supplementary Figure 3: Electrophysiological recordings of iPSC-derived MNs
(A-B) Representative current clamp recordings during ramp depolarization and quantification of single (A, n=110 and n=67 for control and patients, respectively; Mann-Whitney test, **P values is 0.01) or repetitive (B, n=110 and n=67 for control and n=67 for control and patients respectively) action potentials (APs) in mutant FUS expressing MNs and controls indicating that both are similar.
(C-F) Voltage dependent Na + and K + currents elicited upon stepwise depolarization in increments of 10 mV from a holding potential of -70 mV to 40 mV (C, D, n=10 and n=44 for control and patients, respectively). The normalized maximal Na + amplitudes (F, n=10 and n=44 for control and patients, respectively; Mann-Whitney test, ****P values is 0.0001) are significantly lower in mutant FUS patients-derived motor neurons, but K + amplitudes (E, n=110 and n=67 for control and patients, respectively) showed no change.
(B) Quantification using qPCR of the total amount of FUS or knocked-in FUS (with 3×flag) mRNA in H9-hESC and in the different FUS overexpressing hESC lines before and after adding doxycycline (1µg/ml) from day 17 until day 38 of MN differentiation.
(C) Immunostaining of FUS and Neurofilament light chain in inducible hESC lines. Scale bar = 20 µm.

Supplementary Figure 5: No effect of FUS expression on differentiation of hESCs into MNs
(A) Different hESCs containing inducible constructs expressing wild type (wt) FUS or two different mutant FUS constructs (R521H and P525L) integrated into the AAVS1 locus were differentiated into MNs and were stained for choline acetyltransferase (ChaT), Isl1, NFL and Synapsin1in combination with a DAPI staining. In the '+Dox' condition, doxycycline (1µg/ml) was added from day 17 until day 38. Immunostaining of NFL in MNs derived from iPSCs of a healthy control (3/2), an isogenic control (R521R) and a patient (2/2) line. MNs were stained at the 4 th week of differentiation. Scale bar = 20 µm.

Supplementary Figure 7: ELISA detection of phosphatidylcholine levels as a function of differentiation
(A) Phosphatidylcholine levels in culture media of MNs show a decreased trend in patientderived cells. Medium samples were taken for ELISA assays after two days on the culture. Control lines (control 1: 3/2; control 2: 3/3) are shown in blue and patient lines (R521H: 2/2; P525L: 3/1-2) in red.
(B) Phosphatidylcholine level in culture medium of MNs derived from patient iPSCs and isogenic controls with or without an overnight treatment with ACY-738 (1µM).
(C) Phosphatidylcholine level in cultured medium of patient and isogenic control MNs with or without HDAC6 knock down using an ASO.

Supplementary Figure 8: No effect of HDAC6 inhibition on FUS localization in human fibroblasts
Immunostaining for FUS in fibroblasts (3/1 patient carrying the P525L mutation and 3/3 is a healthy control) with or without a treatment with ACY-738 (1µM) or Tubastatin A (1µM). Scale bar= 40 µm.

Supplementary Figure 9: Restoration of ER-mitochondrial overlay and axonal transport by Tubastatin A treatment
(A) Immunostaining for ER (using mouse PDI antibody) and mitochondria (using rabbit TOM-20 antibody) of MNs derived from iPSC from patients carrying the P525L mutation and healthy controls with and without an overnight treatment with Tubastatin A (1 μM). The separate views show co-localized pixels in cell body and neurites. Scale bar = 5 µm.
(B) Quantification of stationary mitochondria, moving mitochondria and ratio between moving to total mitochondria normalized to a neurite length of 100 µm during 200 s from MNs derived from patient and healthy control iPSC lines at the 4 th week after plating with and without a Tubastatin A treatment. (n=10, n=14, n=11, n=13 for Ctr1+DMSO, Ctr1+Tubastatin A, P525L+DMSO and P525L+Tubastatin A respectively. Data are plotted as mean ± SEM; One-way ANOVA with post-hoc Tukey's test; **P values of 0.01 for t-test).
(C) Quantification of stationary ER vesicles, moving ER vesicles and ratio between moving to total vesicles normalized to a neurite length of 100 µm during 200 s from MNs derived from patient and healthy control iPSC lines at the 4 th week after plating before and after Tubastatin A treatment. (n=10, n=16, n=12, n=14 for Ctr1+DMSO, Ctr1+Tubastatin A, P525L+DMSO and P525L+Tubastatin A respectively. Data are plotted as mean ± SEM; One-way ANOVA with post-hoc Tukey's test; *, **P values of 0.05 and 0.01 for t-test, respectively).