DNA instability in postmitotic neurons

Abstract

Huntington's disease (HD) is caused by a CAG repeat expansion that is unstable upon germ-line transmission and exhibits mosaicism in somatic tissues. We show that region-specific CAG repeat mosaicism profiles are conserved between several mouse models of HD and therefore develop in a predetermined manner. Furthermore, we demonstrate that these synchronous, radical changes in CAG repeat size occur in terminally differentiated neurons. In HD this ongoing mutation of the repeat continuously generates genetically distinct neuronal populations in the adult brain of mouse models and HD patients. The neuronal population of the striatum is particularly distinguished by a high rate of CAG repeat allele instability and expression driving the repeat upwards and would be expected to enhance its toxicity. In both mice and humans, neurons are distinguished from nonneuronal cells by expression of MSH3, which provides a permissive environment for genetic instability independent of pathology. The neuronal mutations described here accumulate to generate genetically discrete populations of cells in the absence of selection. This is in contrast to the traditional view in which genetically discrete cellular populations are generated by the sequence of random variation, selection, and clonal proliferation. We are unaware of any previous demonstration that mutations can occur in terminally differentiated neurons and provide a proof of principle that, dependent on a specific set of conditions, functional DNA polymorphisms can be produced in adult neurons.

Fig. 1.

Progression of CAG repeat instability is region-specific and comparable between mice. Each row is a compilation of ABI377 traces representing tissues taken from the same R6/1 mouse. Mosaicism is apparent by 3 months and progressively develops toward expansion with age. The three regions exhibit differing levels of instability: the striatum contains the largest proportion of expansions within its population of CAG repeats; the brainstem has the widest range of expansions; and instability in the cortex is more modest but still detectable. The overall shape of the repeat distribution is conserved between mice for the same brain region with the variance increasing with age. In both striatum and brainstem, the multimodal nature of the radical component of the traces is apparent at 9 months (also see Figs. 2 and 3). x axis, CAG repeat length as indicated; y axis, allele abundance.

Fig. 2.

Comparison of region-specific signatures of DNA instability among three HD mouse models. In both the R6/1 and HdhQ150 models at 9 months of age, the most pronounced mosaicism can be seen in the striatum, olfactory bulb, colliculus, and cerebellum. Although the CAG sizes differ, the overall shape of the traces is preserved. The potential for the development of similar region-specific distributions is apparent in R6/2 mice at 14 weeks of age (end-stage disease) where mosaicism in the striatum and cerebellum can already be observed.

Fig. 3.

Region-specific signatures of DNA instability and allele expression. Shown is a comparison of CAG repeat traces from DNA and RNA that have been extracted from the striata of the same R6/1 mouse at 9 months of age (n = 3). As for DNA, the region-specific RNA traces are consistent between mice. The DNA and RNA CAG repeat ranges are comparable and are composed of a number of peaks indicative of the multimodal nature of the radical component. The position but not the relative height of the peaks is conserved between the DNA and RNA traces. Therefore, the relative RNA allele abundance is the product of mode-specific expression levels. Note that there is no consistent relationship between the repeat length and respective expression level. Blue traces, DNA; red traces, RNA.

Fig. 4.

Differential instability rates in neuronal and nonneuronal cells from human and mouse HD striata. (A) Neuronal and nonneuronal cells in R6/1 striatum were distinguished by NeuN antibody staining and isolated by laser microdissection. The superimposed traces from neuronal and nonneuronal populations display an obvious difference by 6 months. Adult neuronal cells almost exclusively carry somatically expanded alleles. There is greater heterogeneity in the nonneuronal profile, which might reflect the greater heterogeneity of cell types. Green traces, nonneuronal cells; orange traces, neuronal cells. (B) Cell populations were isolated from human patient striata as for mouse, and the CAG repeat distributions were complied by small-pool PCR. The extent of CAG mosaicism is greatest in the neuronal cell populations.

Fig. 5.

MSH3 is present in neuronal cells in the human and mouse striatum. Sections of R6/1 and WT mice aged 9 months and from HD and control human striatum were immunoprobed with antibodies to NeuN to identify neurons and to MSH3. All nuclei were visualized by using TO-PRO-3. MSH3 is predominantly found in neurons in both mouse and human striata. Arrows, neuronal cells; arrowheads, nonneuronal cells. (Scale bar: 20 μm.) (See SI Fig. 8 for larger version).