**2. Results**

#### *2.1. LGF Improves Behavioral Working Memory in APPswe Mice*

The alternance index in the Y-maze, a parameter considered as an indicator of mnemonic function, was significantly reduced in the APPswe mice with respect to WT (Figure 1A). This important marker of working memory was recovered in APP-LGF treated mice (Figure 1A). APP mice also exhibited a reduced number of entries in comparison with WT mice, indicating a low general locomotor activity (18.8 ± 0.9 (*n* = 10) and 11 ± 1.8\* (*n* = 11) in WT and APP mice, respectively \* *p* ≤ 0.05 vs. WT). Instead, no significant differences were observed in the number of entries after LGF treatment in comparison with APP mice (APP-LGF: 13 ± 1.7 (*n* = 12)).

The marble-burying test is a useful model of neophobia, anxiety, and obsessive-compulsive behavior. As shown in Figure 1B, the marble burying test index was significantly reduced in APPswe aged mice, and LGF recovered this index of potential hippocampal affection.

**Figure 1.** Liver growth factor (LGF) improves cognitive behavior in APPswe mice. Panel (**A**) shows how the alternance index in the Y-maze is significantly reduced in APP mice, and how LGF treatment recovers this important marker of working memory. As shown in panel (**B**), the marble burying test index is significantly reduced in APP mice, and LGF recovers this potential index of hippocampal and cortical affection. Results represent the mean ± SEM of 11 to 16 independent mice. The statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. \* *p* ≤ 0.05, \*\*\* *p* ≤ 0.001 vs. wild type (WT). + *p* ≤ 0.01, ++ *p* ≤ 0.01, +++ *p* ≤ 0.001 vs. APP mice.

#### *2.2. LGF Modulates*β*-Amyloid Protein Expression in Hippocampus and Cerebral Cortex of APPswe Mice*

β-amyloid protein accumulation in the Central Nervous System (CNS) is the most important feature of the experimental model of AD used in this study. As shown in Figure 2A, APPswe mice expressed the APP 695 human protein, and several Aβ peptides of different molecular weight lower than 60 kDa which expression was up-regulated in the hippocampus and cerebral cortex, in comparison with WT mice (Figure 2B). The immunohistochemical analysis also revealed the presence of Aβ-positive plaques in the hippocampus (40 <sup>±</sup> 7 (*<sup>n</sup>* <sup>=</sup> 5) Aβ-positive plaques/mm2) and cerebral cortex (38 <sup>±</sup> 6 (*<sup>n</sup>* <sup>=</sup> 5) <sup>A</sup>β-positive plaques/mm2) of APPswe mice. These plaques had different sizes with a 78 <sup>±</sup> 9% of them showing a diameter lower than 25 μm in both brain structures (Figure 2C). In the APP-LGF experimental group, the total number of plaques was not significantly different to that observed in APPswe mice (54.6 <sup>±</sup> 3.2 (*n* = 4) and 44 <sup>±</sup> 3.5 (*n* = 4) Aβ-positive plaques/mm2in the hippocampus and cerebral cortex, respectively). However, LGF treatment significantly reduced Aβ protein levels (Figure 2B), and the number of plaques with a diameter higher than 25 μm compared to APPswe mice treated with the vehicle (Figure 2D). Besides, LGF slightly augmented, but not significantly, augmented the percentage of plaques with a diameter lower than 25 μm by 1.5 ± 0.1-fold and 1.3 ± 0.14-fold in the hippocampus and cerebral cortex, respectively.

#### *2.3. E*ff*ects of LGF in Tau Phosphorylation and Protein Ubiquitination*

A neuropathological feature of AD is the accumulation of Tau and ubiquitin in the neurofibrillary tangles (NFT). At 20 months of age, phospho-Tau/Tau ratio was increased by 2.5-fold in the hippocampus of APPswe mice, and LGF administration partially reduced this parameter to similar values of those observed in WT mice (Figure 3A,D). Phospho-Tau/Tau ratio was similar in the cerebral cortex of

APPswe and WT mice, but LGF treatment significantly reduced this ratio below WT levels (Figure 3A). A similar effect was observed in the cerebral cortex of WT-LGF treated mice (Figure 3A).

Protein ubiquitination is essential in an important number of processes including protein degradation by the proteosome. Accumulation of polyubiquitinated proteins was observed in the hippocampus and cerebral cortex of APPswe mice, and LGF administration reduced this parameter in both structures (Figure 3B,D).

HSP-70 is a chaperone that allows the correct folding of mis-folded proteins that could lead to their aggregation. As shown in Figure 3C, the hippocampus of APP-LGF treated mice showed significantly higher levels of HSP-70 than WT or APPswe mice. HSP-70 protein expression was down-regulated in the cerebral cortex of APPswe mice, and LGF treatment restored its levels to control values (Figure 3C,D).

**Figure 2.** LGF reduces amyloid-β accumulation and the size of amyloid-β positive plaques in the hippocampus and cerebral cortex of APPswe mice. Panels (**A**,**B**) show a Western blot representative image of Aβ aggregates in the hippocampus (**A**) and the quantification of Aβ-aggregates ≤40 kDa in the hippocampus and cerebral cortex (**B**). Note that Aβ protein levels are up-regulatedin the hippocampus and cerebral cortex of APPswe mice and that LGF treatment significantly reduces its expression in both structures. Panel (**C**) shows a representative image of Aβ-positive plaques of large (**C**, black arrows) and small size (**C**, black arrowheads) in one hemisphere of APP mice brain (scale bar: 600 μm). Note that in APP-LGF mice, large plaques are reduced, while small plaques increase. Panel (**D**) shows how LGF treatment significantly reduces the number of plaques with a diameter higher than 25 μm in the hippocampus and cerebral cortex. Results represent the mean ± SEM of 7 to 8 (**B**) and 4 to 5 (**D**) independent mice in each experimental group. The statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. \*\*\* *p* ≤ 0.001 vs. WT. + *p* ≤ 0.05, +++ *p* ≤ 0.001 vs. APP mice.
