Increased cell proliferation and neurogenesis in the adult human Huntington's disease brain

Abstract

Neurogenesis has recently been observed in the adult human brain, suggesting the possibility of endogenous neural repair. However, the augmentation of neurogenesis in the adult human brain in response to neuronal cell loss has not been demonstrated. This study was undertaken to investigate whether neurogenesis occurs in the subependymal layer (SEL) adjacent to the caudate nucleus in the human brain in response to neurodegeneration of the caudate nucleus in Huntington's disease (HD). Postmortem control and HD human brain tissue were examined by using the cell cycle marker proliferating cell nuclear antigen (PCNA), the neuronal marker beta III-tubulin, and the glial cell marker glial fibrillary acidic protein (GFAP). We observed a significant increase in cell proliferation in the SEL in HD compared with control brains. Within the HD group, the degree of cell proliferation increased with pathological severity and increasing CAG repeats in the HD gene. Most importantly, PCNA+ cells were shown to coexpress beta III-tubulin or GFAP, demonstrating the generation of neurons and glial cells in the SEL of the diseased human brain. Our results provide evidence of increased progenitor cell proliferation and neurogenesis in the diseased adult human brain and further indicate the regenerative potential of the human brain.

Fig. 1.

PCNA immunoreactivity is increased in the subependymal layer in HD cases. (a) Table summarizing the age, postmortem delay, pathological grade, CAG trinucleotide repeat length in the IT15 gene, and PCNA grade for each HD case is shown. For each HD case, the level of PCNA immunoreactivity in the subependymal layer was assessed by using a qualitative five-point grading scale (PCNA grade). (b) Compared with the control brain, the thickness of the subependymal layer (SEL) and the number of PCNA⁺ cells in the SEL are increased as the pathological grade of HD increases (grade 1 to grade 3). In each case, the boundary of the SEL with the caudate nucleus (CN) is indicated by an arrow. (EP, ependymal layer; LV, lateral ventricle). (c) Graph showing a significant correlation between pathological grade and PCNA grade in the HD cases (Spearman rank correlation, r = 0.8972, P ≤ 0.003). (d) Graph showing a significant correlation between CAG trinucleotide repeat length in the IT15 gene and PCNA grade in the HD cases (Spearman rank correlation, r = 0.8168, P ≤ 0.02). (Scale bar, 80 μm.)

Fig. 2.

Western immunoblotting with the PCNA antibody using human control (three cases) and HD (four cases) SEL homogenates demonstrates the specificity of the PCNA antibody by a single strong band at 36 kDa.

Fig. 3.

PCNA⁺, βIII-tubulin⁺, and GFAP⁺ cells are located in different regions within the SEL as demonstrated with bright-field immunohistochemical serial sections through the EP, SEL, and CN of a grade 2 HD brain. (a) PCNA⁺ cells are evenly distributed within the SEL (arrows demonstrate examples of PCNA⁺ cell bodies), and the EP is also densely stained. (b) βIII-tubulin⁺ fibers and cell bodies are present in the lower part of the SEL (arrows indicate cell bodies with immunoreactive fibers). (c) GFAP⁺ cells are distributed homogenously throughout the SEL and in the CN (arrows indicate examples of GFAP cell bodies).

Fig. 4.

Newly generated cells in the SEL of the HD brain exhibit either a neuronal or glial phenotype. (a) Confocal microscopy demonstrates that the SEL of the HD brain contains newly generated cells that coexpress PCNA and βIII-tubulin and stain with a Hoechst stain. The new neurons are located in the lower part of the SEL. (Scale bar, 6 μm.) (b–d) Higher magnification of the neuron, which is outlined with a box in a. (Scale bar, 3 μm.) (b) Hoechst (blue) stains the nucleus of cells in the SEL. (c) PCNA (red) labels the nucleus of a new cell and has a granular appearance (19). (d) βIII-tubulin (green) labels the cytoplasm of neurons early in their development. (e) The merged image demonstrates coexpression of PCNA and βIII-tubulin in the same cell that displays a Hoechst⁺ stain, indicating that neurogenesis occurs in the SEL of the HD brain. (f) Confocal microscopy demonstrates that the SEL of the HD brain also contains newly generated cells that coexpress PCNA and GFAP and stain with a Hoechst stain. These new glial cells are located in the upper part of the SEL in the HD brain. (Scale bar, 18 μm.) (g–i) Higher magnification of the glial cell, which is outlined with a box in f. (Scale bar, 9 μm.) (g) Hoechst (blue) stains the nucleus of cells in the SEL. (h) PCNA (red) labels the nucleus of the cell with punctate staining. (i) The astrocytic marker GFAP (green) labels the cytoplasm of the cell. (j) The merged image demonstrates coexpression of PCNA and GFAP in the same cell that displays a Hoechst stain, indicating the occurrence of gliogenesis. Images a–e and f–j were each obtained by using different excitation wavelengths, and the signal was detected at different emission wavelengths.