During our initial characterization of the tet-off APP transgenic lines in 2005 we noted motor hyperactivity during both light and dark phases of the light cycle that was severe enough to preclude cognitive analysis . Here we show that this motor hyperactivity can be substantially attenuated by delaying overexpression of the mutant protein until adulthood. This finding suggests that the postnatal brain is susceptible to APP overexpression in a way that the mature brain is not. Exposure to transgenic APP during postnatal development had long-lasting effects on adult brain function that were exacerbated by continued expression of mutant APP. However, motor hyperactivity in juvenile-onset mice persisted at a modest level even after therapeutic suppression of transgenic APP, indicating that once established, the behavior is no longer dependent on continued APP overexpression to be maintained.
Our findings are consistent with past reports describing motor hyperactivity in several other lines of APP overexpressing mice. Significant elevations in open field activity and home cage ambulation have been described in both Tg2576 and CRND8 models [9, 13, 19–22]. Both of these transgenic lines are controlled by the prion protein promoter [2, 5], which in other transgenic models is active in the brain before birth . As with the CaMKIIα promoter used here, overexpression of transgenic APP from the prion protein promoter would be expected by the second postnatal week when the nigrostriatal pathway undergoes considerable structural maturation . Endogenous APP plays a role in shaping neuronal morphology and establishing synaptic connections during development, and loss of APP through targeted deletion in vivo or by shRNA knockdown in vitro results in neurite overgrowth and excessive, but poorly aligned, pre- and post-synaptic structures [40–43], reviewed in . Paradoxically, overexpression of APP can cause similar changes in immature neurons, increasing neurite outgrowth and the density of dendritic spines [45, 46]. In vivo, the effect of APP overproduction on neuron morphology is often confounded by the concurrent accumulation of Aβ in models expressing FAD mutations. However, overexpression of wild-type APP – absent the Aβ overproduction of FAD variants – leads to increased synapse density both in the brain and in the periphery [47–49]. Taken together, changes in APP expression (both up and down) can affect neuronal outgrowth and synapse formation in the brain and would be present at the right time and place to modify neural circuitry in the maturing nigrostriatal pathway.
While delaying the onset of transgenic APP until adulthood reduces the severity of motor hyperactivity, it does not completely eliminate the behavior. Slight but significant increases in ambulation developed by 7 wk of APP overexpression and persisted at the same level when tested at 4 mo. However, unlike the residual hyperactivity that remained following therapeutic APP suppression in mice with juvenile transgene expression, 1 mo of dox treatment completely normalized motor activity in adult-onset mice. This indicates that in adult-onset mice, motor hyperactivity was dependent on the presence of transgenic APP. Consistent with this, past work has shown that infusion of Aβ into the brains of wild-type mice can acutely modulate dopamine release [50, 51]. However, the initial lag between induction of transgenic APP by 1 wk after dox withdrawal (Figure 5) and the appearance of hyperactivity at 7 wk (Figure 3) suggests that the behavioral phenotype was not a direct effect of APP overproduction. Instead, the delay between transgene induction and the appearance of hyperactivity suggests that the effect may be indirect, perhaps through functional changes in the nigrostriatal pathway, such as modulation of dopamine receptor sensitivity, alterations in dopamine production, or transmitter uptake. Consistent with this possibility, APP/PS1 transgenic mice show altered tyrosine hydroxylase immunoreactivity and diminished size of dopaminergic neurons within the susbstantia nigra long before the appearance of amyloid . Alternatively, the delay between transgene induction and behavioral changes in the adult-onset mice (i.e., activity after 1 wk of APP over-expression was identical to NTG but by 7 wk became significantly elevated) may suggest dependence on the accumulation of an aggregated, but reversible, form of Aβ. Further experiments, such as in vivo microdialysis to measure striatal dopamine release, or secretase inhibition to prevent Aβ production during continued APP expression, will be needed to distinguish these possibilities.
Our study further demonstrates that the motor phenotype can be modulated independently from amyloid pathology. Hyperactivity began prior to amyloid formation, in both juvenile- and adult-onset mice. In both groups, cortical plaque burden was < 0.05% after 2 mo of APP overexpression, yet ambulation was already elevated a week earlier at 7 wk. Hyperactivity worsened after amyloid formation in juvenile-onset mice, but remained unchanged from pre-deposit levels in adult-onset animals. The dissociation between plaques and motor activity was most pronounced following therapeutic suppression of transgenic APP. Dox treatment begun after the appearance of amyloid deposits has been shown to stabilize plaque burden, preventing further accumulation but not promoting clearance [35, 53]. APP suppression completely normalized motor activity in adult-onset mice and significantly reduced hyperactivity in juvenile-onset animals, despite the continued presence of amyloid in both.
There are three caveats to our interpretation that are worth noting. The first is that as late as 2 mo after onset, the level of transgene expression in dox-reared animals is not identical to dox-naïve. Although the level of APP overexpression following adult-onset ultimately matches that of juvenile-onset mice, it starts off approximately 25% lower. If the absolute level of transgenic APP and not the age at which it is expressed is the critical factor in determining hyperactivity, we may have mistaken a concentration difference for an effect of timing. That other APP transgenic models with lower expression levels also display motor hyperactivity suggests this is not the case. There is likely a minimum level of mutant APP expression needed to evoke this phenotype, as a companion line of tetO-APPswe/ind mice generated at the same time as the line used here, but which produced approximately one-quarter the amount of transgenic APP, did not develop motor hyperactivity (line 70, data not shown). The adult-onset mice studied here expressed well in excess of this minimum, but the fact that they expressed at lower levels for several months and started almost a week later than juvenile-onset animals (once embryonic expression and the delay between dox withdrawal and transgene onset are taken into account) meant that they also developed amyloid at a later age. If the absolute amyloid levels were a critical factor in determining hyperactivity, a better experimental design would have matched adult- and juvenile-onset animals for plaque burden rather than duration of APP expression. In most of our comparisons, amyloid load was lower in adult-onset animals (0.26 ± 0.038% vs. 1.6 ± 0.18% after 4 mo of transgenic APP expression, 2.92 ± 0.49% vs. 9.31 ± 0.67% at 6 mo). However, under conditions that matched amyloid load rather than duration of APP overexpression (14.3% at 8 mo after adult onset (calculated), and 9.3% at 6 mo after juvenile onset (actual)), hyperactivity was still greater in mice that expressed APP from birth. This leads into the final caveat to our interpretation of the data. In several of our experiments, the magnitude of the difference in open field ambulation and ambulation is driven by a small number of outliers. While the size of the effect shrinks when these animals are removed from analysis, the significance remains. Thus, the behavioral impact of transgenic APP during postnatal development is fully penetrant, albeit highly variable in scale.