Conserved pharmacological rescue of hereditary spastic paraplegia-related phenotypes across model organisms

Abstract

Hereditary spastic paraplegias (HSPs) are a group of neurodegenerative diseases causing progressive gait dysfunction. Over 50 genes have now been associated with HSP. Despite the recent explosion in genetic knowledge, HSP remains without pharmacological treatment. Loss-of-function mutation of the SPAST gene, also known as SPG4, is the most common cause of HSP in patients. SPAST is conserved across animal species and regulates microtubule dynamics. Recent studies have shown that it also modulates endoplasmic reticulum (ER) stress. Here, utilizing null SPAST homologues in C. elegans, Drosophila and zebrafish, we tested FDA-approved compounds known to modulate ER stress in order to ameliorate locomotor phenotypes associated with HSP. We found that locomotor defects found in all of our spastin models could be partially rescued by phenazine, methylene blue, N-acetyl-cysteine, guanabenz and salubrinal. In addition, we show that established biomarkers of ER stress levels correlated with improved locomotor activity upon treatment across model organisms. Our results provide insights into biomarkers and novel therapeutic avenues for HSP.

Figure 1.

spas-1 mutation caused age-dependent HSP-related motor phenotype in C. elegans. (A) Wild-type N2 nematodes showed no clear paralysis phenotype until the age of Day 12 of adulthood. spas-1(ok1608) and spas-1(tm683) strains displayed a progressive paralysis phenotype. (B) RT-PCR analysis revealed a loss of expression of the spas-1 gene in the spas-1(ok1608) nematodes.

Figure 2.

Methylene blue, guanabenz, salubrinal and phenazine prevented the HSP-related motor phenotype in spas-1 mutation in C. elegans. 60 μM methylene blue (MB), 50 µm guanabenz (Gua), 50 salubrinal (Sal) and 25 μm phenazine (Phe) reduced the % of paralysis in spas-1(ok1608) worms.

Figure 3.

Methylene blue, guanabenz, salubrinal and phenazine extended the lifespan of spas-1 mutant in C. elegans. (A) Methylene blue (MB), (B) guanabenz (Gua) and (C) salubrinal (Sal), (D) but not phenazine (Phe), prolonged the lifespan in spas-1(ok1608) mutants in C. elegans.

Figure 4.

Oxidative stress is associated with the paralysis observed in the spas-1 mutants, and methylene blue, guanabenz, salubrinal and phenazine prevent this oxidative stress in spas-1 mutant nematodes. (A) 10 mm N-Acetyl-L-cysteine (NAC), a strong antioxidant, reduced the % of paralysis in the spas-1(ok1608) nematodes. (B) 2′,7′-dichlorofluorescein diacetate (DCF-DA) fluorescent photos and (C) quantification of spas-1(ok1608) worms after treatment with methylene blue (MB), guanabenz (Gua), salubrinal (Sal) and phenazine (Phe). **P < 0.01 versus N2 controls; ^#P < 0.05, ^##P < 0.01 versus spas-1(ok1608) nematodes (Student's t-tests). Silencing spas-1 induces the ER-related hsp-4/BiP expression, and methylene blue, guanabenz, salubrinal and phenazine prevent the hsp-4 increase in C. elegans. (D) Representation and (E) quantification of spas-1 RNAi which induces the hsp-4/BiP expression in the zcIs4[hsp-4::GFP] worms, but exposure to methylene blue (MB), guanabenz (Gua), salubrinal (Sal) and phenazine (Phe) prevent this hsp-4::GFP increase. *P < 0.05, **P < 0.01 versus untreated empty vector (EV) controls, ^###P < 0.001 versus untreated spas-1 RNAi (Student's t-tests).

Figure 5.

Methylene blue rescues the locomotor defects caused by spastin loss-of-function in Drosophila. (A) Flies expressing pan-neuronal RNAi against spastin display poor climbing performance (N = 6) which is rescued with overnight Methylene blue treatment. (B) Significantly increased climbing performance is observed at 2 min with spastin RNAi transgenic flies treated with Methylene blue (P ≤ 0.01, t-test). (C) Transheterozygous spastin mutants (spastin^5-75/spastin^17-7) display poor climbing performance (N = 7) which is rescued with overnight Methylene blue treatment. (D) Significantly increased climbing performance is observed at 2 min with transheterozygous flies treated with Methylene blue (P ≤ 0.001, t-test).

Figure 6.

Phenazine rescues the locomotor defects caused by spastin loss-of-function in Drosophila. (A) Flies expressing pan-neuronal RNAi against spastin display poor climbing performance (N = 10) which is rescued with overnight phenazine treatment. (B) Significantly increased climbing performance is observed at 2 min with spastin RNAi transgenic flies treated with phenazine (P ≤ 0.01, t-test). (C) Transheterozygous spastin loss-of-function mutants (spastin^5-75/spastin^17-7) display poor climbing performance (N = 10) which is rescued with overnight phenazine treatment. (D) Significantly increased climbing performance is observed at 2 min with transheterozygous flies treated with methylene blue (P ≤ 0.01, t-test).

Figure 7.

N-Acetyl-L-cysteine rescues the locomotor defects caused by spastin loss-of-function in Drosophila. (A) Flies expressing pan-neuronal RNAi against spastin display poor climbing performance (N = 6) which is rescued with overnight N-Acetyl-L-cysteine treatment. (B) Significantly increased climbing performance is observed at 2 min with spastin RNAi transgenic flies treated with N-Acetyl-L-cysteine (P ≤ 0.05, t-test). (C) Transheterozygous spastin mutants (spastin^5-75/spastin^17-7) display poor climbing performance (N = 8) which is rescued with overnight N-Acetyl-L-cysteine treatment. (D) Significantly increased climbing performance is observed at 2 min with transheterozygous flies treated with N-Acetyl-L-cysteine (P ≤ 0.01, t-test).

Figure 8.

Methylene blue rescues exaggerated oxidative stress caused by spastin loss-of-function in Drosophila. Representative confocal images of BiP immunostaining in (A) wild-type, (B) spastin RNAi expressing flies treated with vehicle and (C) spastin RNAi flies treated with methylene blue. (D) Flies expressing pan-neuronal RNAi against spastin display elevated BiP level compared with wild-type flies. This level is rescued to normal level by the administration of methylene blue provided in the negative geotaxis experiments (N = 5, P < 0.01, t-test).

Figure 9.

Methylene blue, guanabenz, salubrinal and phenazine partially rescue the morphological phenotype and reduce microtubule defects of the zebrafish spastin morphant. (A) Zebrafish embryos at 30 hours post fertilization (hpf). spastin morphant embryos show abnormal morphological features compared with embryo injected with control morpholino. Arrows show defects in the head, yolk sac extension and tail. These defects are partially rescued by 60 μm methylene blue (MB), 20 μm guanabenz (Gua), 20 μm salubrinal (Sal) and 20 μm phenazine (Phe). Scale bar is 500 μm. (B) Quantification of morphological defects shown in (A). (C) Whole-mount immunohistochemistry of 30 hpf zebrafish embryos labeled with a microtubule marker. A lateral view of the trunk is shown, with the rostral side of the embryo to the left. spastin morphants show a disorganized spinal cord, with thin microtubules at the level of the motor neuron axons. These defects are partially rescued by 60 μm methylene blue (MB), 20 μm guanabenz (Gua), 20 μm salubrinal (Sal) and 20 μm phenazine (Phe). Scale bar is 30 μm, MO: morpholino. (D) Zebrafish embryos injected with a control morpholino show very low level of fluorescence when treated with 2′,7′-dichlorofluorescein diacetate (DCF-DA) (a biomarker for oxidative stress) compared with embryos injected with a morpholino against spastin. Methylene blue, guanabenz, salubrinal and phenazine reduced the fluorescence. (E) Quantification of fluorescence in DCF-DA-treated embryos show a significant reduction of fluorescence upon treatment with the 4 drugs (*P < 0.001 versus control morphant embryos; ^#P < 0.01 versus spastin morphant embryos).