Reduced acetylated α-tubulin in SPAST hereditary spastic paraplegia patient PBMCs

Abstract

HSP-SPAST is the most common form of hereditary spastic paraplegia (HSP), a neurodegenerative disease causing lower limb spasticity. Previous studies using HSP-SPAST patient-derived induced pluripotent stem cell cortical neurons have shown that patient neurons have reduced levels of acetylated α-tubulin, a form of stabilized microtubules, leading to a chain of downstream effects eventuating in increased vulnerability to axonal degeneration. Noscapine treatment rescued these downstream effects by restoring the levels of acetylated α-tubulin in patient neurons. Here we show that HSP-SPAST patient non-neuronal cells, peripheral blood mononuclear cells (PBMCs), also have the disease-associated effect of reduced levels of acetylated α-tubulin. Evaluation of multiple PBMC subtypes showed that patient T cell lymphocytes had reduced levels of acetylated α-tubulin. T cells make up to 80% of all PBMCs and likely contributed to the effect of reduced acetylated α-tubulin levels seen in overall PBMCs. We further showed that mouse administered orally with increasing concentrations of noscapine exhibited a dose-dependent increase of noscapine levels and acetylated α-tubulin in the brain. A similar effect of noscapine treatment is anticipated in HSP-SPAST patients. To measure acetylated α-tubulin levels, we used a homogeneous time resolved fluorescence technology-based assay. This assay was sensitive to noscapine-induced changes in acetylated α-tubulin levels in multiple sample types. The assay is high throughput and uses nano-molar protein concentrations, making it an ideal assay for evaluation of noscapine-induced changes in acetylated α-tubulin levels. This study shows that HSP-SPAST patient PBMCs exhibit disease-associated effects. This finding can help expedite the drug discovery and testing process.

Keywords: biomarkers; hereditary spastic paraplegia; microtubule; neurodegenerative disease; noscapine.

Figure 1

Acetylated α-tubulin levels in SPAST HSP patient and control PBMCs. Flow cytometry was used to measure acetylated α-tubulin in overall PBMCs and PBMC cell sub-types. (A) The forward-scatter and side-scatter (FSC/SSC) gated plots represent the total mononuclear cells. (B) Live PBMCs were identified using a cell viability dye. Viable cells have a relatively lower fluorescence level of the viability dye and vice versa for non-viable cells. Gating was applied to identify the proportion of viable cells. (C) CD2, CD19, and CD14 cell surface antibodies were used to identify PBMC cell sub-types T cells, B cells and monocytes. The scatter plot shows CD14 monocytes and CD19 B cells separated using the surface antibodies. The proportion of live PBMCs (D) and the proportion of T cells (E), B cells (F) and monocytes (G) is shown for all HSP-SPAST patients and healthy controls. (H–J) Levels of acetylated α-tubulin in PBMCs were measured in patient and control PBMC samples. Scatter plot of an acetylated α-tubulin negative control sample (H) and representative control (I) and patient (J) samples are shown. (K) Histogram plot showing acetylated α-tubulin levels in representative patient and control PBMC samples. (L) Patient and control group level comparison based on acetylated α-tubulin geometric mean fluorescence intensity in all PBMCs. (M) Histogram plot showing acetylated α-tubulin levels in representative patient and control T cells. Patient and control group level comparison of T cells (N), B cells (O) and monocytes (P) based acetylated α-tubulin geometric mean fluorescence intensity. Mean ± SEM.

Figure 2

Plasma and brain noscapine levels in noscapine treated mouse. Mouse was orally administered with increasing concentrations of noscapine: 100, 200, and 400 mg/kg. Noscapine was measured in mouse plasma (A) and brain homogenate (B) at 60 min post-dose using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). N = 4 mouse per group. One way ANOVA confirmed a significant noscapine treatment effect in the mouse plasma (p < 0.01) and brain (p < 0.01). Tukey’s post-hoc multiple comparisons showed group differences (^*p < 0.05 and ^**p < 0.01). Mean ± SEM.

Figure 3

Acetylated α-tubulin levels in noscapine treated mouse. Acetylated α-tubulin was measured in the brain of the mouse dosed with noscapine. Mouse was orally administered with increasing concentrations of noscapine: 100, 200, and 400 mg/kg. One way ANOVA confirmed a significant noscapine treatment effect on acetylated α-tubulin in the mouse brain (p < 0.01). Tukey’s post-hoc multiple comparisons showed group differences (^*p < 0.05, ^**p < 0.01, ^***p < 0.001). N = 3 or 4 mouse per group. a.u. = arbitrary unit. Mean ± SEM.

Figure 4

Acetylated α-tubulin levels in noscapine treated human PBMCs. Acetylated α-tubulin was measured in human PBMCs treated with noscapine (in vitro) at 10 μM for 1 h. Student’s t-test confirmed a significant noscapine treatment effect on acetylated α-tubulin levels (^****p < 0.0001). N = 8 PBMC samples per untreated and noscapine treated groups. Mean ± SEM.