Doxy ameliorates motor performance
Motor deficits are core manifestations in PD. Gait instability and muscle weakness significantly affect the daily living of patients who constantly experience a disabling fear of falling .
To evaluate the effect of Doxy treatment on gait instability and muscle weakness, NTG and A53T mice, receiving either Veh or Doxy, were tested in the beam walk and paw grip test. As shown in Fig. 1A and B, A53T+Veh-treated mice were significantly impaired in their gait stability experiencing a higher number of footslips and spending a longer time in performing the task than Veh- and Doxy-NTG animals. Notably, Doxy significantly improved the gait stability in A53T mice which performed significantly better than A53T+Veh mice. A53T+Doxy-treated mice, indeed, displayed a significant reduction of both the number of footslips and the time spent for performing the task compared to Veh-treated Tg animals. Besides an effect on mouse gait stability, Doxy treatment also significantly ameliorated muscle strength in A53T mice. In fact, A53T+Doxy-treated animals showed a longer latency in the paw grip test compared to the Veh-treated Tg group (Fig. 1C).
Doxy restores cognitive functions and daily life activity in A53T mice
PD patients also develop deficits in memory and lose their ability to execute daily life activities. To assess the effects of the Doxy treatment at cognitive level, IP treated A53T and NTG mice were tested in both NORT and nesting test starting from treatment day 27. We found that, while A53T+Veh-treated mice showed a significant memory impairment, spending an equal time investigating the novel and familiar object, Doxy completely restored memory in A53T mice which spent a longer time on the novel object rather than on the familiar one, showing both a higher % of investigation time on the novel object and a discrimination index (DI) comparable with the control condition (Fig. 2A, B). Doxy also significantly ameliorated the daily life activity measured through the nesting test in A53T mice. As shown in Figure 2C and D, Doxy rescued the nesting behavior of A53T mice 24 and 48 hours after the nest presentation. At 48 hours the nesting score of Doxy-treated Tg mice was comparable to both NTG experimental groups.
Doxy prevents cortical and hippocampal neuronal death in A53T mice
Since Doxy treatment restored cognitive functions in A53T mice, we sought whether such a positive outcome was associated with a Doxy-induced neuronal protection in several brain areas involved in cognitive functions, such as the pre-frontal cortex (PFC), neo-cortex (neoCTX) and hippocampus (HP). NeuN staining of both the PFC and the neoCTX demonstrated that Doxy counteracts neuronal loss in A53T mice. Indeed, through the quantification of NeuN+ cell density, a slight but significant reduction was observed in the PFC and neoCTX of A53T+Veh mice, whereas a neuronal density comparable to NTG+Veh- and NTG+Doxy-treated mice was found in the same brain regions in A53T mice receiving Doxy (Fig. 3A-C).
Aside from a neuroprotective effect of Doxy in cortical regions, we found that it also promoted neuronal survival in the HP. As shown in Figure 3D, we found a significant reduction of NeuN+ cell density in the hippocampal CA3 subfield of A53+Veh-treated mice when compared to both Veh- and Doxy-treated NTG mice. In contrast, neuronal loss did not occur in A53T+Doxy treated animals as confirmed by the quantitative analysis of NeuN+ cell density (Fig. 3E).
Doxy reduces α-syn oligomers in A53T mice
As aforementioned α-syn deposits are typical neuropathological hallmarks of PD. Thus, through an immunohistochemical approach we investigated whether functional effects of Doxy were associated with changes in deposited α-syn. As shown in Fig 4A, we found that Doxy did not affect α-syn deposition both in the CTX and HP of A53T mice, which was confirmed through a quantitative analysis (Fig 4B and C).
Among α-syn assemblies, oligomers have been pointed out as the higher neurotoxic moieties capable of affecting neuronal survival, as well as cognitive functions and synaptic plasticity [3-6, 8, 9, 32]. To investigate whether Doxy treatment affected α-synO production in A53T mice, we performed western blotting analyses for α-syn in the Triton X-100 soluble and insoluble fraction of both CTX and HP from A53T+Veh- and A53T+Doxy-treated mice. While not significant differences were detected in the amount of α-syn monomer and oligomers in the cortical and hippocampal soluble fraction (data not shown), we found differences in the insoluble one. In particular, while both in the CTX (Fig. 4D and E) and HP (Fig 3G and H) of A53T mice Doxy did not affect the α-syn monomer protein levels, the levels of oligomeric assemblies were significantly reduced in both brain areas.
Glial cell activation is reduced by Doxy treatment in A53T mice
Neuroinflammation is a key factor fostering PD as well as cognitive deficits [6, 9, 10, 33-37]. α-synOs have been previously reported to trigger glial activation , and it is well-known that Doxy exerts pleiotropic activities such as inhibition of α-syn aggregation and gliosis [20, 38]. To assess whether the recovery of A53T cognitive functions was linked to a Doxy-mediated reduction in neuroinflammation, we addressed the extent of glial activation in the neoCTX and HP of A53T mice. As shown in Fig. 5A and Fig. 5D, the extent of either microglial (IBA1) or astroglial (GFAP) cell activation was decreased in both area upon Doxy treatment. Indeed, in both areas A53T+Veh-treated mice displayed microglia and astrocytes with an enlarged soma, whereas in A53T mice receiving Doxy these cells showed a resting morphology with a smaller soma and a lower number of branches. To further define the extent of gliosis, we quantified the % of IBA1- and GFAP-marked area in neocortical and hippocampal area among experimental groups. In the neoCTX, Doxy completely abolished microglial cell activation, indeed the % of IBA1-marked area was significantly reduced in Doxy-treated A53T mice compared to A53T receiving Veh, and similar to either Veh- or Doxy-treated NTG mice (Fig. 5B). Similar to microglial activation, the quantitative analysis of the % of GFAP-marked area was significantly reduced in Doxy treated A53T mice compared to Veh-treated Tg animals. Moreover, Veh-treated A53T mice had a larger neocortical area occupied by GFAP+ cells compared to Veh and Doxy treated NTG mice (Fig. 5C).
We also examined the degree of gliosis in HP, a crucial brain region involved both in long term memory and daily life activity [39 ,40]. As shown in Fig. 4E Doxy treatment significantly reduced the % of IBA1-marked area in A53T+Doxy mice compared to A53T+Veh ones, which had a significantly higher % of IBA1-marked area than Veh- or Doxy-treated NTG mice (Fig. 5E).
Our data also highlight a inhibitory effect of Doxy on astroglial cells in the HP of A53T mice (Fig. 4F). In fact, while the hippocampal area occupied by GFAP+ cells was significantly larger in A53T+Veh-treated mice, compared to either untreated or treated NTG mice, it was significantly smaller in Doxy-treated A53T mice.
Striatal inflammation commonly occurs in PD  and it has been also associated with motivation and motor impairment . Thus, we assessed gliosis in the striatum of either Veh or Doxy-treated NTG and A53T mice. While microglial activation was significantly hampered in A53T+Doxy-treated mice compared to Veh-treated A53T animals (Fig 5A and B), astroglial cell activation was not affected (Fig. 6A and C).
Doxy restores hippocampal LTP in association with inhibition of proinflammatory mediators, and an increased expression of BDNF
Synaptic plasticity, functionally assessed through the LTP, is a key neuronal mechanism underling learning and new memory formation.
To investigate whether Doxy treatment was capable of restoring synaptic plasticity in A53T mice, hippocampal LTP was measured in NTG and A53T mice after 30 days of treatment with Veh or 10 mg/Kg of Doxy. As shown in Figure 5D-F, a significant impairment of LTP in A53T mice receiving Veh was completely rescued by Doxy treatment. In A53T+Doxy-treated mice LTP amplitude was comparable to NTG receiving either Veh or Doxy (Fig 7A). Notably, the Doxy treatment did not affect LTP in NTG mice (Fig. 7A-C), further confirming the absence of potential adverse effects.
Proinflammatory cytokines and neurotrophic factors can influence neuronal functions and LTP. In particular, increased levels of IL-1β, IL-6 and TNF-α, as well as the reduced expression of the neurotrophic factor BDNF, can inhibit LTP [43-49]. Since we have found that A53T mice showed an impaired LTP and Doxy was able to restore it, we have investigated in A53T mice whether Doxy could affect the level of IL-1β, IL-6, TNF-α and BDNF in the hippocampus of Tg compared to NTG mice. Our analysis revealed that IL-1β expression (Fig. 7C) was significantly higher in A53T+Veh-treated mice compared to NTG animals receiving Veh, and that Doxy treatment significantly reduced its expression. Consistent with the results obtained for IL-1β expression in the hippocampus, we found a significantly higher expression of IL-6 and TNF-α in Veh-treated A53T mice compared to the NTG ones, and Doxy significantly dampened also their expression (Fig 7D, F)
It has been recently demonstrated that Doxy favors the expression of the neurotrophic factor BDNF  which is involved in memory processing as well as LTP. Thus, we assessed BDNF expression in A53T mice. As shown in Fig. 7G, while the expression of BDNF was significantly lower in A53T+Veh compared to NTG+Veh mice, in A53T mice receiving Doxy, it was comparable to the level of NTG+Veh mice.