Memory deficits, gait ataxia and neuronal loss in the hippocampus and cerebellum in mice that are heterozygous for Pur-alpha

Abstract

Pur-alpha is a highly conserved sequence-specific DNA and RNA binding protein with established roles in DNA replication, RNA translation, cell cycle regulation, and maintenance of neuronal differentiation. Prior studies have shown that mice lacking Pur-alpha (-/-) display decreased neurogenesis and impaired neuronal differentiation. We sought to examine for the first time, the behavioral phenotype and brain histopathology of mice that are heterozygous (+/-) for Pur-alpha. Standardized behavioral phenotyping revealed a decreased escape response to touch, limb and abdominal hypotonia, and gait abnormalities in heterozygous Pur-alpha (+/-) mice, compared to wild-type (+/+) littermates. Footprint pattern analyses showed wider-based steps, increased missteps and more outwardly rotated hindpaws in heterozygous Pur-alpha (+/-) mice, suggestive of cerebellar pathology. The Barnes maze and novel object location testing revealed significant memory deficits in heterozygous Pur-alpha mice, suggestive of hippocampal pathology. Quantitative immunohistochemical assays of the vermal region of the cerebellum and CA1-3 regions of the hippocampus revealed reduced numbers of neurons in general, as well as reduced numbers of Pur-alpha+-immunopositive neurons and dendrites in heterozygous Pur-alpha mice, compared to wild-type littermates. Past studies have implicated mutations in Pur-alpha in several diseases of brain development and neurodegeneration. When combined with these new findings, the Pur-alpha heterozygous knockout mice may provide an animal model to study mechanisms of and treatments for Pur-alpha-related cognitive deficiencies and neuropathology.

Keywords: PURA; animals; brain pathology; knockout; mice; puralpha.

Figure 1

Select results (neural reflexes and open field activities) from a general health screen. (A) Several neural reflexes were decreased in heterozygous Pur-alpha (+/−) mice (both sexes mixed), compared to wild-type (+/+) littermates (both sexes mixed) (two-way ANOVA showed a difference between the genotypes, p<0.0001). Posthoc analysis showed a significantly decreased escape behavior, limb tone and abdominal tone in the heterozygous mice, as well as a trend towards a reduction in acoustic startle to two different sudden noises. (B, C) Similar results were observed after dividing the groups by sex. (D–F) In the open field assay, both male and female heterozygous Pur-alpha (+/−) mice showed similar amounts of time spent in the perimeter versus the center of the arena and similar mean speeds, yet increased gait abnormalities. *p<0.05, compared to wild-type (+/+) littermates. Mean ± SEM shown.

Figure 2

Gait abnormalities were evident in heterozygous Pur-alpha (+/−) mice during forward walking. (A) Representative figures show that heterozygous Pur-alpha (+/−) mice have more foot drags, missteps, outwardly positioned hindpaws, and wider-based steps, compared qualitatively to wild-type (+/+) mice. (B) Footprint pattern analysis showed that hindpaws of heterozygous Pur-alpha mice were more rotated than wild-type (+/+) littermates (specifically, hindpaws showed an increased angle during walking so that they pointed away from the forward direction of the mouse). (C) Footprint pattern analysis also showed that heterozygous Pur-alpha mice had wider fore base and hind base gaits, and less overlap between forepaws and hindpaws during forward walking. **p<0.01, compared to wild-type (+/+) littermates. Mean ± SEM shown.

Figure 3

Assessment of memory abilities in novel object location and novel object recognition assays in wild-type mice (+/+) and heterozygous Pur-alpha (+/−) mice. (A) Cartoons showing the design of the novel object recognition assay of the location of three objects in an open field chamber. Mice were allowed to explore the location of 3 objects during a 3 minute acquisition assay. For the retention test that occurred 1 hour later, two objects were moved and one object remained in the same location. (B) Both male and female heterozygous Pur-alpha mice showed similar decreases in percent time exploring moved objects relative to unmoved objects, compared to age-matched male and female wild-type (+/+) littermates. This is indicative of a deficit in spatial location memory in the heterozygous Pur-alpha mice. (C) Neither group showed deficits in novel object recognition. *p<0.05, compared to wild-type (+/+) littermates. Mean ± SEM shown.

Figure 4

Assessment of memory abilities in a Barnes maze test at 24 hours after the last day of acquisition training. (A) Heterozygous Pur-alpha (+/−) mice showed increased numbers of visits to several blank holes (*:p<0.05), compared to age-matched wild-type (+/+) littermates. *p<0.05, compared to wild-type (+/+) littermates. Mean ± SEM shown.

Figure 5

Further assessment of memory abilities in Barnes maze tests. Animals were trained for 4 days to learn the maze (4 trials/day for a total of 16 trials) before being tested in retention tests at 24 hours (day 5 of testing) and 30 days after the last day of acquisition training. (A) Heterozygous Pur-alpha (+/−) mice showed slower average velocity around the Barnes maze on the last day of acquisition training, and at both retention test time points, than wild-type (+/+) littermates. (B,C) Heterozygous Pur-alpha mice showed longer total distance traveled and greater distance prior to their head entering the target hole, at both retention test time points. (D) Heterozygous Pur-alpha mice had shorter distances prior to their heads entering a blank hole during the last day of acquisition training and 24 hour (day 5) retention test. (E) Heterozygous Pur-alpha mice had an overall longer latency before the head entered the target hole during the 24 hour (day 5) retention test. (F) Representative cartoons showing that wild-type (+/+) mice had a higher prevalence for a direct spatial search strategy as shown on left, while heterozygous Pur-alpha mice showed more random, serial, or a mix (as shown on right) during the Day 5 Retention Test. *:p<0.05 and **p<0.01, compared to wild-type (+/+) littermates. Mean ± SEM shown.

Figure 6

Reduced Pur-alpha immunostained neurons and dendrites in hippocampus and cerebellum of mature (11 months of age) heterozygous Pur-alpha (+/−) mice, compared qualitatively to wild-type (+/+) mice littermates. (A–D). Low power and higher power photomicrographs from hippocampal (Hippoc) CA2 and CA3 regions and vermal cerebellum (Cbl) region of wild-type (+/+) mice. Insets in A and B show neurons with cytoplasm intensely stained for Pur-alpha as well as long intensely stained dendrites. (E–H) Low power and higher power photomicrographs from hippocampal (Hippoc) CA2 and CA3 regions and cerebellum and vermal cerebellum (Cbl) region of heterozygous Pur-alpha (+/−) littermates. Arrows in the insets of panels E and H indicate hippocampal neurons and Purkinje cells lacking Pur-alpha staining, respectively. Panel F and its inset shows that neurons in the CA3 region of a heterozygous Pur-alpha mouse have little to no Pur-alpha stained dendrites. Scale bars in A, C, E and G = 100 micrometers; Scale bars in B, D, F and H = 25 micrometers.

Fig. 7

Reduced numbers of Pur-alpha immunostained neurons and dendrites in hippocampus, cerebellum and amygdala of mature (11 months of age) heterozygous Pur-alpha (+/−) mice, compared to wild-type (+/+) mice littermates. (A) Estimated total number of Purkinje cell bodies, Pur-alpha immunostained (+) Purkinje cell bodies, and Pur-alpha + Purkinje cell dendrites in the cerebellum. (B–D) Estimated total number of neuronal cell bodies, Pur-alpha-immunopositive neuronal cell bodies, and Pur-alpha-immunopositive dendritic elements in the hippocampus CA1-3 region, amygdala and prefrontal cortex. Results of two-way ANOVA shown at upper right in each panel; significant posthoc findings shown as *p<0.05 and **p<0.01, compared to wild-type (+/+) littermates. Mean ± SEM shown.