Our documentation of motor impairment, behavioral perturbations, altered neurochemical function, and the development of synucleinopathy and tauopathy over the adult lifespan of A53T mice provides a novel view of these processes in a well-studied PD model. Our analysis has clarified the relationship between the motor phenotype of these mice and the apparent loss of anxiety-like behavior, suggesting that the latter is largely a consequence of or at least concomitant with reduced locomotion. In addition, the results of our study illustrate two phenomena critical to our understanding of how the Syn proteins function in both normal and pathological circumstances.
First, we provide evidence that a change in the local concentration of α-Syn, β-Syn, and γ-Syn can be associated with functional modulation of DAT in the mouse brain. In cellular models of DAT trafficking, A53T α-Syn binds poorly to DAT and fails to modulate DAT trafficking to the cell surface 
. Similarly, DA uptake and DAT distribution to striatal membranes are increased in young A53T mice where bioavailability of abundant A53T α-Syn is highest. This supports the view that the loss of α-Syn modulation of DAT through over-expression of trafficking incompetent A53T α-Syn results in increased localization of DAT to the cell surface. Uptake of DA through DAT was normalized in older animals where bioavailability of A53T α-Syn was reduced and expression of β-Syn and γ-Syn was increased. Indeed, we have observed recently that all three wild-type Syn proteins have a similar capacity to modulate DAT distribution (unpublished data). The normalization of DA uptake with aging may therefore relate to a shift in modulation of DAT from α-Syn to the other Syn proteins β-Syn and γ-Syn. The progressive removal of trafficking-incompetent A53T α-Syn and replacement with β-Syn and γ-Syn may also indicate a compensatory response involved in the restoration of normal DAT trafficking. The effects on NET of A53T α-Syn have not been examined previously, and though α-Syn-NET and α-Syn-DAT interactions are mediated by the same NAC region 
, these studies produced no conclusive result regarding the modulation of NET by A53T α-Syn.
Second, we propose that mis-trafficking of DAT in the presence of excess A53T α-Syn contributes to biochemical alterations in the striatum, including increased levels of hyper-phosphorylated Tau (p-Tau). We have shown that over-expression of trafficking-incompetent A53T α-Syn in young mice leads to increased DAT distribution to the cell surface and elevated intracellular DA exposure. Subsequent to these changes in DA uptake we observe increased activation of JNK and Tau hyperphosphorylation at several JNK-targeted epitopes. Importantly, phosphorylation at the PHF-1 epitope and other p-Tau sites in the striatum appeared to resolve after 8 months, which correlated with the restoration of DA uptake to normal levels. Tau phosphorylation returned to normal levels at all but one site in 12 month-old A53T mice, indicating that p-Tau accumulation is transient and not likely to be contributing to alterations in DAT trafficking or function. Thus, despite significant loss of SNpc neurons and severe synucleinopathy known to occur throughout the brain stem and spinal cord 
, striatal structural integrity appears to have been largely preserved. The transient nature of increased Tau phosphorylation and lack of striatal pathology suggests that observed changes in endogenous Syn protein expression at later ages could have restored Syn-dependent DAT trafficking and therefore be involved in alleviation of DA-induced stress kinase signaling.
Modulation of DAT trafficking through a direct interaction with α-Syn was initially described over ten years ago 
, and subsequent work has identified several potential mechanisms that could contribute to trafficking of DAT by α-Syn 
as well as the other Syn family proteins β-Syn and γ-Syn (unpublished data). While modeling of Syn-DAT trafficking in mammalian cell culture has been informative, prior studies have been unsuccessful in detecting Syn modulation of DAT, with most reporting negative findings 
. These results therefore constitute the first evidence for a dynamic relationship between Syn protein concentration and DAT function in the mouse brain. Of particular interest is the correlation between excess DA uptake, stress kinase activation, increased Tau phosphorylation, and changes in Syn expression. It is currently unknown whether these events are coordinated, and the mechanisms involved have not been identified. A proposed compensatory response involving increased expression of Syn family members β-Syn and γ-Syn is consistent with evidence for redundancy between the three Syn proteins that is regulated at the transcriptional level 
, and indicates the need for further investigation into the mechanisms that control Syn gene expression.
Here, we have attempted to overlay changes in the integrity and function of the nigrostriatal pathway with age-dependent changes in anxiety-like and depressive-like behavior, as well as accumulating motor dysfunction. Perhaps the most consistent behavioral feature of A53T mice was a reduced level of locomotor activity which was evident on varied analyses from several different tests. This is consistent with increased clearance of DA from striatal synapses, although increased uptake of DA is not likely to be the only factor involved in the reduced activity. To provide context, it should be considered that DAT over-expressing mice have increased striatal DA clearance of a similar magnitude, yet no change in basal locomotor activity 
. This suggests that the reduced activity level of the A53T α-Syn mice is only partially dependent on the increase in DA uptake. Furthermore, the enhancement of striatal DA uptake fades with aging, yet locomotor activity remains low, and motor strength and endurance are additionally lost. A related mouse model of A53T α-Syn over-expression with a similar reduction in locomotor activity 
also exhibited robust changes in both pre-synaptic and post-synaptic dopaminergic function including increased striatal DA content, enhanced expression of DA receptors, and reduced expression of DA metabolizing enzymes 
. This reflects a circumstance where DA neurotransmission is impaired, resulting in sensitization of the synapses. These changes are consistent with increased DA uptake capacity, but suggest that DA release probability, a measure of the rate of DA vesicle fusion with the synaptic membrane, may also be reduced in the presence of excess α-Syn 
. Accumulation with aging of both β-Syn and γ-Syn, though likely to normalize DAT trafficking as proposed here, could have the additional effect of exacerbating the perturbation of DA release, and thus may simultaneously contribute to the overall lowered activity level.
The decreased immobility scores on the FST in older A53T mice are more difficult to align with what is known about the reduced dopaminergic function and motor activity in these animals. The FST primarily measures the behavioral response to entrapment, with swimming and climbing behaviors viewed as an attempt to escape, while immobility is viewed as an indication of stress-induced despair or depression 
. It is important to note that while less active in the OFT and EPM, A53T mice were more active in the FST, with reduced immobility scores (increased swimming) at 4 and 8 months. The pattern of reduced depressive-like behavior diverged dramatically at 12 months, as symptomatic A53T mice (WHT latency<20 s) adopted an immobile posture throughout the FST period, while asymptomatic A53T animals maintained lower immobility scores compared to WT. The increase in FST immobility scores in symptomatic A53T mice suggests that increased depressive-like behaviors observed in other rodent models of PD (primarily neurotoxin models) may be linked to the acute loss of dopaminergic tissues and development of motor impairment rather than the development of pre-motor symptoms analogous to the co-morbidity of depression with PD (for review see 
). Successfully modeling the constellation of non-motor symptoms that accompany PD, especially those that typically occur prior to clinical diagnosis, remains a pressing goal for pre-clinical researchers that could provide a means for testing prophylactic treatments or other therapeutic measures 
. While the A53T mouse lines are considered one of the more successful models of PD and synucleinopathy 
, the observed reductions in anxiety-like and depressive-like behaviors are essentially opposite what would be predicted based on clinical reports. In addition, some reports show that striatal DA concentration may actually be elevated in a similar A53T mouse model 
, a finding that is clearly at odds with the accepted pathophysiology of PD 
. Also, administration of DA or L-DOPA fails to restore normal electrophysiologal properties to striatal neurons in A53T mice 
. Taken together with our work here, these studies suggest that A53T mice may not be ideal for modeling the full range of PD-associated symptoms, or at least that only limited inferences should be drawn from A53T mouse models with regard to the progression of sporadic PD.
In contrast, the vesicular monoamine transporter 2 deficient mouse (VMAT2 LO) undergoes a more gradual loss of monoamine storage capacity that is associated with increased depressive-like behavior in aged VMAT2 LO animals 
. Younger VMAT2 LO mice have FST immobility scores similar to WT, suggesting that their later increase in depressive-like behavior is linked to the progressive loss of monoamine function, including NE and serotonin in addition to DA. This type of functional loss is only partially reproduced in A53T mice, as a decrease in SNpc neurons was not accompanied by a loss of striatal MSN dendritic spine density. Previously, there was limited reported evidence of nigrostriatal degeneration in aging A53T α-Syn mice 
, with investigations focused primarily on severe synucleinopathy and motor neuron losses in the spinal cord 
. More recently, we have reported that TH-expressing neurons in the SN of A53T α-Syn mice were reduced by 30% 
. A similar result has been reproduced here, where a more thorough analysis found a substantial (~20%) and statistically significant loss of both total and TH-expressing SNpc neurons in aged A53T α-Syn mice. These results argue against the prior consensus that dopaminergic pathology is absent in A53T α-Syn mice. An analysis of striatal medium spiny neurons (MSN), however, showed that dendritic spine density was unaltered as late as ten months in A53T α-Syn mice. While this presents difficulties for a clear description of the dopaminergic status of these animals, the apparent sparing of MSN spine density is not necessarily inconsistent with a partial loss of dopaminergic innervation. MSN make up 95% of striatal neurons, and are subdivided into several distinct classes that are nonetheless indistinguishable in terms of morphology or anatomical distribution 
. Recent work has shown that in a toxin-based mouse model of parkinsonism the MSN sub-type expressing the D2 dopamine receptor undergoes selective dendritic spine loss, while neighboring MSN expressing the D1 dopamine receptor are largely unaffected 
. Similar works showing substantial loss of MSN dendritic spine density typically involve lesions causing SNpc neuron loss much greater than 20% 
, and so it is possible that the limited loss of TH-expressing cells in the present study is insufficient to produce significant dendritic spine remodeling. Furthermore, MSN sub-types were not differentiated here; future work should examine both D1 and D2 dopamine receptor expressing cells to determine whether these functionally distinct populations are differentially affected in the A53T α-Syn mouse. Further analysis of striatal function seems especially needed given the evidence presented here and elsewhere 
that these animals undergo SNpc cell losses. Furthermore, we have shown that the biochemical phenotype of A53T α-Syn mice includes excess cytosolic DA exposure, and the time course of these changes parallel the behavioral phenotype of these mice. We propose that the function of increased Tau phosphorylation in this context is more complex, being a marker of DA-induced stress kinase activation in the initial stages that fades as DAT modulation by β-Syn and γ-Syn is restored. These findings support our view of the central importance of a linkage between Tau hyper-phosphorylation and the development of synucleinopathy in dopaminergic tissues, including the progression of PD