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. 2015 Sep 15;24(18):5285-98.
doi: 10.1093/hmg/ddv248. Epub 2015 Jun 29.

Analysis of YFP(J16)-R6/2 Reporter Mice and Postmortem Brains Reveals Early Pathology and Increased Vulnerability of Callosal Axons in Huntington's Disease

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Free PMC article

Analysis of YFP(J16)-R6/2 Reporter Mice and Postmortem Brains Reveals Early Pathology and Increased Vulnerability of Callosal Axons in Huntington's Disease

Rodolfo G Gatto et al. Hum Mol Genet. .
Free PMC article

Abstract

Cumulative evidence indicates that the onset and severity of Huntington's disease (HD) symptoms correlate with connectivity deficits involving specific neuronal populations within cortical and basal ganglia circuits. Brain imaging studies and pathological reports further associated these deficits with alterations in cerebral white matter structure and axonal pathology. However, whether axonopathy represents an early pathogenic event or an epiphenomenon in HD remains unknown, nor is clear the identity of specific neuronal populations affected. To directly evaluate early axonal abnormalities in the context of HD in vivo, we bred transgenic YFP(J16) with R6/2 mice, a widely used HD model. Diffusion tensor imaging and fluorescence microscopy studies revealed a marked degeneration of callosal axons long before the onset of motor symptoms. Accordingly, a significant fraction of YFP-positive cortical neurons in YFP(J16) mice cortex were identified as callosal projection neurons. Callosal axon pathology progressively worsened with age and was influenced by polyglutamine tract length in mutant huntingtin (mhtt). Degenerating axons were dissociated from microscopically visible mhtt aggregates and did not result from loss of cortical neurons. Interestingly, other axonal populations were mildly or not affected, suggesting differential vulnerability to mhtt toxicity. Validating these results, increased vulnerability of callosal axons was documented in the brains of HD patients. Observations here provide a structural basis for the alterations in cerebral white matter structure widely reported in HD patients. Collectively, our data demonstrate a dying-back pattern of degeneration for cortical projection neurons affected in HD, suggesting that axons represent an early and potentially critical target for mhtt toxicity.

Figures

Figure 1.
Figure 1.
Analysis of YFP expression in the CCX of YFP(H) and YFP(J16) transgenic mice. Confocal microscopic images (10×) showing portions of the CCX, the CC and the ST illustrate major differences in the pattern and levels of YFP fluorescence between YFP(H) (A) and YFP(J16) mice brains (B). At higher magnification (25×), confocal images show that YFP expression in the CCX of YFP(H) mice is limited to a subset of corticospinal neurons located in layer V (C). In contrast, the pattern of YFP expression is much broader in the CCX of YFP(J16) mice, also comprising cortical neurons in layers III–VI (D). In YFP(H) mice, nearly all YFP-positive cortical neurons (pseudo-colored in green) co-localized with the corticospinal projection neuron marker Ctip2 (arrows) (E), but only few co-localized with the callosal projection neuron marker SATB2 (in red) (F). Conversely, little YFP-Ctip2 co-localization was observed in the CCX of YFP(J16) mice (G). Instead, most YFP-positive neurons in these mice co-localized with SATB2 (H), indicating that an important fraction of YFP-positive cortical neurons in the CCX of YFP(J16) mice corresponds to callosal projection neurons. Scale bars: 100 µM (A, B), 20 µM (C–H).
Figure 2.
Figure 2.
YFP(J16)-R6/2(160Q) mice feature presymptomatic alterations in CC integrity. Diffusion tensor color maps of YFP(J16) (A) and YFP(J16)-R6/2(160Q) mice (B) at presymptomatic age P30 revealed structural alterations in the CC (white arrows). (C) Quantitative data showed reduced mFA values for the CC of YFP(J16)-R6/2(160Q) mice, compared with YFP(J16) mice (*P < 0.05; n = 3 per experimental group). (D) A confocal microscopy image of the CC of YFP(J16) mice shows a homogeneous pattern of YFP fluorescence (yellow), a neat parallel organization of callosal axons and an aligned distribution of 4',6-diamidino-2-phenylindole-stained callosal glia nuclei (blue). (E) In contrast, some areas of the CC showed reduced levels of YFP fluorescence (delineated by dashed lines) in YFP(J16)-R6/2(160Q) mice, and callosal axons appear less organized overall. In addition, callosal glia nuclei displayed a more scattered distribution and abnormal morphology, compared with that of YFP(J16) mice. Scale bars in D–E: 10 µM.
Figure 3.
Figure 3.
Age-dependent degeneration of callosal axons in YFP(J16)-R6/2(160Q) mice. (A) In YFP(J16) mice, confocal microscopic images of the CC showed even levels of YFP fluorescence and a consistent pattern of callosal axon organization from ages P30 to P90. (B) In contrast, YFP fluorescence levels were reduced, and the fibrillar pattern of YFP fluorescence was gradually lost as YFP(J16)-R6/2(160Q) mice aged. By age P90, callosal axons appeared severely degenerated. Axonal swellings (white arrows), which were observed at P90 in YFP(J16) mice, become conspicuous at P30 in YFP(J16)-R6/2(160Q) mice. (D) Consistent with the pathophysiology of HD, axonal degeneration was less pronounced in YFP(J16)-R6/2(120Q) mice than in YFP(J16)-R6/2(160Q) mice. (C) Quantitative data show YFP fluorescence levels for YFP(J16) mice (control, solid gray bars, n = 3), YFP(J16)-R6/2(120Q) mice (120Q, vertical dashed bars, n = 3) and YFP(J16)-R6/2(160Q) (160Q, horizontal dashed bars, n = 3). *P < 0.05, **P < 0.01. Scale bar: 10 µM.
Figure 4.
Figure 4.
Reductions in YFP fluorescence levels in YFP(J16)-R6/2(160Q) mice reflect axonal pathology. (A) In coronal brain sections, SMI-31 antibody against neurofilament heavy chain subunits shows a fibrillar staining pattern for callosal axons of YFP(J16) mice up to age P90. (B) But in YFP(J16)-R6/2(160Q) mice, this fibrillar pattern is lost in an age-dependent manner, much as seen for YFP fluorescence (compare with Fig. 3A and B). (C) Confocal images show YFP fluorescence in sagittal sections of the CC at age P90. Compared with YFP(J16) mice, YFP(J16)-R6/2(160Q) mice displayed a marked reduction in YFP fluorescence levels, and an apparent reduction in the caliber of YFP-positive axons. (D) EM images of the CC confirmed this observation, thus validating YFP fluorescence levels as a reporter of axonal integrity. (E) The mean caliber of callosal axons was significantly reduced in YFP(J16)-R6/2(160Q) mice, compared with YFP(J16) mice (n = 3 mice, 1500 axons per experimental group); ***P < 0.001. All scale bars are 10 µM.
Figure 5.
Figure 5.
Callosal axon degeneration does not result from loss of neuronal cell bodies. (A) Representative confocal microscopic images (40×) of YFP(J16) and YFP(J16)-R6/2(160Q) mice CCX (layers III–V) at ages P30, P60 and P90. A marked reduction in YFP fluorescence levels was observed for the cortical neuropil (for example, the areas delimited by dashed lines) at all the ages analyzed. (B) Quantitation of YFP-positive neuronal cell bodies revealed similar numbers for YFP(J16) (control, solid gray bars, n = 3) and YFP(J16)-R6/2(160Q) mice (160Q, horizontal dashed bars, n = 3) at all the ages analyzed. Scale bar: 10 µM.
Figure 6.
Figure 6.
Mutant huntingtin aggregates are spatially and temporally segregated from degenerating callosal axons. Merged confocal images obtained from YFP(J16)-R6/2(160Q) mice show YFP fluorescence (pseudo-colored in green) and mhtt immunoreactivity (in red), as revealed by EM48 monoclonal antibody. (A) At presymptomatic age P30, a few mhtt aggregates (arrowheads) were observed in the CCX. Although signs of callosal axon pathology were evident at this age (see Fig. 3B), no mhtt aggregates were observed in the CC. (B) At age P90, abundant nuclear (long arrows) and neuritic (short arrows) mhtt aggregates were found in the CCX, but only occasional ones were found in the CC (short arrows). All scale bars: 10 µM.
Figure 7.
Figure 7.
Differential vulnerability of axons in YFP(J16)-R6/2(160Q) mice. Confocal microscopic images (63×) show YFP-positive axons within the ONT (A), the lateral funicular region of the spinal cord (B) and sciatic nerves (C) of YFP(J16) and YFP(J16)-R6/2(160Q) mice at symptomatic age P60. Plots in A'–C' show YFP fluorescence levels for these white matter structures at ages P30, P60 and P90 (n = 3 per genotype). Unlike the CC (see Fig. 3), axons within the ONT and the sciatic nerve show similar YFP fluorescence levels for both experimental groups at all the ages analyzed. However, a small decrease was observed for spinal cord axons at late symptomatic age P90 (*P < 0.05). Scale bars: 10 µM.
Figure 8.
Figure 8.
Increased vulnerability of callosal axons in HD brains. (A) Representative images corresponding to coronal and sagittal sections of the CC's genu show reduced SMI-31 immunoreactivity in the CC of human HD cases, compared with controls (n = 3 per group, cases 45 and 85 are shown, see Table 1 for case demographics), suggesting a marked loss of callosal axons in HD. (B) In contrast, the pattern of SMI-31 immunoreactivity in the ONT remained largely unchanged. (C) Quantitative immunofluorescence data showed reduced ratios of callosal (CC) to ONT SMI-31 immunoreactivity (IR) in HD brains, compared with control cases (*P < 0.05). (D) A Pearson's test shows a statistically significant correlation between mhtt polyQ tract length and CC/OT SMI-31 immunoreactivity ratios for HD cases analyzed in this study (r = –0.85). Scale bar: 50 µM.

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