H-ABC tubulinopathy revealed by label-free second harmonic generation microscopy

Abstract

Hypomyelination with atrophy of the basal ganglia and cerebellum is a recently described tubulinopathy caused by a mutation in the tubulin beta 4a isoform, expressed in oligodendrocytes. The taiep rat is the only spontaneous tubulin beta 4a mutant available for the study of this pathology. We aimed to identify the effects of the tubulin mutation on freshly collected, unstained samples of the central white matter of taiep rats using second harmonic generation microscopy. Cytoskeletal differences between the central white matter of taiep rats and control animals were found. Nonlinear emissions from the processes and somata of oligodendrocytes in tubulin beta 4a mutant rats were consistently detected, in the shape of elongated structures and cell-like bodies, which were never detected in the controls. This signal represents the second harmonic trademark of the disease. The tissue was also fluorescently labeled and analyzed to corroborate the origin of the nonlinear signal. Besides enabling the description of structural and molecular aspects of H-ABC, our data open the door to the diagnostic use of nonlinear optics in the study of neurodegenerative diseases, with the additional advantage of a label-free approach that preserves tissue morphology and vitality.

Figure 1

The supernumerary microtubules in mutant oligodendrocytes could emit detectable second harmonic signal. (A) Schematic setup of the commercial microscope used for imaging. NDD: non-descanned, PMT: Photomultiplier NA: Numerical aperture. (B) The healthy nervous system does not generate detectable second harmonic signal. (C) The pathologic white matter of the taiep rat emits second harmonic signal.

Figure 2

The corpus callosum of taiep rats hosts cells that emit second harmonic signals. (A) Location of the imaged and analyzed region of the CC. (B–D and E–G) Representative SHG micrographs from the CC of a WT and a TUBB4A mutant rat, respectively; Z-stacks of five images, each spaced 7 μm, were generated. In (B) and (E) the sum projections of the five planes are shown, to better appreciate the continuity of the emitting structures in the corpus callosum. All animals tested gave identical results. In (C) and (F) only one plane is shown. Images (D) and (G) are high magnification images from squares in (C) and (F), respectively. Arrow, cell bodies; dotted arrow, elongated structures. (H) Gray values plotted from the line scans crossing over the fibers in the CC of WT and taiep rats; line and image sources are shown just below. (I) Gray level histogram obtained from regions of interest (ROIs) in the CC of the rats (26 + 16 images from 3 taiep and 2 WT rats). (J, K) Gray values of surface profiles; intensities obtained from elongated structures in demyelinated regions are higher than those found in similar locations in healthy rats. The calibration bar in (E) is valid for (B, C) and (F), and the calibration bar in (D) is also valid for (G), with brighter colors representing the highest signal intensity. The direction of laser polarization for SHG microscopy coincides with the scale bar orientation. The average power used for SHG imaging was 13 mW.

Figure 3

Cells that emit second harmonic signal are located in hypomyelinated regions of taiep rats. (A, B) SHG images from an acute preparation of the CC of a 10 months control and mutant rat, respectively. (D, E) Confocal images of fluorescently labeled myelin (red), neurofilaments (green), and nuclei (blue) in the CC of WT (D) and TUBB4A mutant rat (E). The membrane-associated signal appears more intense and organized in control rats than in the mutants and the signal from neurofilaments is less intense in control rats. (C) and (F) Fluorescence of neurofilaments (green dots) and myelin (red dots), (4 WT and 4 taiep rats, *p < 0.05, p = 0.03, two-tailed Mann–Whitney test). The direction of laser polarization for SHG microscopy coincides with the scale bar orientation. The average power used for SHG imaging was 13 mW.

Figure 4

Demyelinated cerebellar white matter in taiep rats has second harmonic generating cells and a higher fluorescence intensity from neurofilaments. (A–F) Representative SHG micrographs from the cerebellum of WT (A–C) and mutant (D–F) rats. All animals tested gave identical results. The images are maximum projections from 5 planes separated by 7 μm. (G–L) Confocal images of stained neurofilaments from the cerebellum of WT (G–I) and mutant rats (J–L). (B, E, H and K) are zoomed regions in the molecular layer and (C, F, I, and L), in the cerebellar white matter, where the difference in signal distribution and intensity is better appreciated. In (F), arrow, cell bodies; star, intense rounded structure and dotted arrow, fiber-like structure. The calibration bar in panel (D), with brighter colors corresponding to the highest signal intensities, is also valid for (A, C and F) while the calibration bar in (E) is also valid for (B). The direction of laser polarization for SHG microscopy coincides with the scale bar orientation. (M) Schematic of cerebellar folia. (N, O) Representative line graphs of the intensity levels detected along the lines shown in panels A and D for SHG images, G and J for NF fluorescence, respectively. In SHG images, the WM of taiep rats shows the highest gray level values of the three layers. The mutant rat also displays higher WM NF fluorescence than the fluorescence from granular and molecular layers (n = 3 TUBB4A rats and 3 WT). (P) Gray levels histograms obtained from second harmonic signal of the cerebellar layers (2 WT and 3 taiep rats). (Q) NF fluorescence intensity in the WM of mutant rats was higher than the fluorescence from control rats, no significant difference was found in molecular and granular layers between WT and taiep (n = 3 taiep and 3 WT rats, *p < 0.05, two-tailed Mann–Whitney test). The average power used for SHG imaging was 13 mW.

Figure 5

In cerebellar white matter, differences in cellular numbers between taiep and WT are more pronounced. (A–D) More nuclei are observed in the corpus callosum and cerebellar WM of taiep rats than in WT. (E, F) Tubulin staining in the corpus callosum of WT (E) and taiep rats (F); inset in (F) shows a magnification of the region pointed by the arrow. (G, H) Cerebellar WM and granular layer in WT (G) and taiep rats (H), arrows in (H) point at cell bodies heavily stained for tubulin. (I-L) Sections labeled with the mature oligodendrocyte marker CC1 in red, and tubulin in green; corpus callosum of WT (I) and taiep rats (J), cerebellar WM of WT (K) and taiep rats (L). The arrow in (J) points to a cell positive for tubulin and the arrow in (L) points to a cell positive for tubulin and CC1. (M) Quantification of DAPI, tubulin and CC1 positive cells/mm² in the corpus callosum and in the cerebellar white matter. Significant differences were found between genotypes for DAPI (+) cells (F_(1,8) = 9.35, p = 0.00028) and for tubulin (+) cells (F_(1,8) = 37.389, p = 0.015), (n = 3 WT and 3 taiep rats, two-way ANOVA followed by Tukey's multiple comparison test was performed. Different letters indicate significant differences between groups).

Figure 6

Differences between taiep and WT white matter come from microtubules. (A, B) Acute tissue sections labeled with SiR-Tubulin. In cerebellar white matter of WT rats no cell bodies were observed (A, 2 WT rats aged 2 months). In contrast, cell bodies and processes labeled for tubulin were detected in taiep rats WM (B, 2 taiep rats aged 8 months). (C, D) SHG images from white matter of WT and taiep rats (2 WT and 2 taiep rats aged 6 months). The average power used for SHG imaging was 20 mW.