*2.4. LGF Modulates Microglia Activation and Reduces Inflammation in APPswe Mice*

Recent reports suggest that microglia play an important role in the evolution of AD. The immunohistochemical analysis of the hippocampus and cerebral cortex of APPswe mice showed that the number of microglial cells that expressed the Ionized Calcium Binding Protein-1 (Iba1) was significantly higher than in WT control mice (Figure 4A). Most of the Iba1-positive cells associated with the plaques were located around them (Figure 4B), and a few showed Iba1-positive processes

penetrating into the plaques (Figure 4C). Iba1 protein levels were also up-regulated in both structures (Figure 4D,H), suggesting the presence of activated microglia in these mice. Increased proliferation has also been associated with microglia activation. As shown in Figure 4E, the expression of PCNA was significantly increased in the hippocampus and cerebral cortex of APPswe mice. Similar to this finding, BrdU incorporation was higher in these mice than in the WT group in both structures (Figure 4F), and more than 61 ± 4% (*n* = 8) of the proliferating cells were Iba1-positive microglia (Figure 4G).

**Figure 3.** Effects of LGF in tau pathology, ubiquitinated protein levels and HSP70 heat shock protein expression. Panel (**A**), shows the relative optical density of phospho-tau/tau ratio analyzed by Western blot and expressed as % vs. WT. Note how LGF treatment reduces the expression of phosphorylated tau in the hippocampus and cerebral cortex in APP mice. Panels (**B**,**C**) show the quantification of accumulated ubiquitinated proteins (**B**) and HSP-70 (**C**) in the hippocampus and cerebral cortex of APPswe mice. Note how LGF reduces the accumulation of ubiquitinated proteins (**B**) and increases HSP70 expression (**C**). Panel (**D**) shows representative Western blot images of phospho-tau, total tau, Hsp-70 and β-actin as charge control. Results represent the mean ± SEM of 4 to 8 independent mice. The statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. \* *p* ≤ 0.05, \*\* *p* ≤ 0.01, \*\*\* *p* ≤ 0.001 vs. WT. + *p* ≤ 0.05, ++ *p* ≤ 0.01, +++ *p* ≤ 0.001 vs. APPswe mice.

The APP-LGF group of animals showed a significant reduction in Iba1 (Figure 4D,H) and PCNA protein expression (Figure 4E,H), and a slight but reduced number of BrdU-positive cells in comparison with APPswe mice (Figure 4F). Besides, LGF treatment increased from 6 ± 2.9 (*n* = 3) to 19 ± 11 (*n* = 4) the percentage of Iba1-positive cells, which prolongations penetrated profoundly in the plaques in the hippocampus.

**Figure 4.** Liver growth factor modulates microglia cell activity in the hippocampus and cerebral cortex of APPswe mice. Panel (**A**) shows the quantification of Iba1-positive microglia cells in APPswe mice. Panels (**B**,**C**) show different responses of Iba1-positive cells: in (**B**) microglial prolongations do not penetrate inside the deposit, while in (**C**) they actively phagocytize the plaque (scale bar: 50 μm). Panels (**D**,**E**) show quantitative Western blot analysis of Iba1 (**D**) and PCNA (**E**) protein expression. Note how LGF administration significantly reduces the over-expression of Iba1 (**D**) and PCNA (**E**) in the hippocampus and cerebral cortex. LGF also reduces BrdU cell incorporation (**F**) as an index of cellular proliferation. Note how BrdU (**G**, red) is associated with Iba1-positive cells (**G**, green, white arrows). Panel (**H**) show representative Western blots of Iba1 and PCNA and their respective β-actin as charge control. The results represent the mean ± SEM of 4 (**A**,**F**) and 8 to 10 (**D**,**E**). independent mice. The statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. \* *p* ≤ 0.05, \*\* *p* ≤ 0.01, \*\*\* *p* ≤ 0.001 vs. WT. + *p* ≤ 0.05, ++ *p* ≤ 0.01, +++ *p* ≤ 0.001 vs. APPswe mice.

GFAP is a protein expressed by astrocytes for which up-regulation has been associated with inflammatory states in chronic processes as AD and aging. As shown in Figure 5A, GFAP was over-expressed in the hippocampus and cerebral cortex of APPswe mice, and LGF treatment significantly reduced its levels in both structures. On the other hand, LGF decreased ASC protein levels that were up-regulated in the hippocampus and the cerebral cortex of APPswe aged mice (Figure 5B,D). ASC is involved in the production of Interleukin-1beta (Il-1beta) but, neither APP nor APP-LGF treated mice showed any significant change in the levels of this pro-inflammatory cytokine in the hippocampus (113 ± 6 (*n* = 6) and 101 ± 7 (*n* = 6) % of IL-1beta expression vs. WT in APP and APP-LGF treated mice, respectively) and the cerebral cortex (102 ± 11 (*n* = 5) and 92 ± 12 (*n* = 6) % of IL-1beta expression vs. WT in APP and APP-LGF treated mice, respectively). Similar results were observed when we analyzed the potential effects of LGF in Tumor Necrosis Factor-alpha (TNF-alpha) protein expression in the hippocampus (92 ± 6 (*n* = 6) and 88 ± 10 (*n* = 8) % of TNF-alpha expression vs. WT in APP and APP-LGF treated mice, respectively) and cerebral cortex (95 ± 6 (*n* = 5) and 107 ± 9 (*n* = 5) % of TNF-alpha expression vs. WT in APP and APP-LGF treated mice, respectively). This latter cytokine mediates LGF-induced neuroregeneration/neuroprotection [6,20].

LGF also modulated the expression of the transcription factor Nrf2, and the class B scavenger receptor CD36, two proteins that regulate oxidative stress and inflammatory responses in AD. As shown in Figure 5D, Nrf2 protein levels were reduced in the hippocampus and cerebral cortex of APPswe mice, and LGF treatment up-regulated Nrf2 protein expression to reach control values. LGF treatment reduced the levels of CD36 that is over-expressed in the hippocampus of APPswe mice (Figure 5C,E). Besides, LGF up-regulated CD36 protein expression in the hippocampus of WT mice (Figure 5C,E), but it lacked activity in the cerebral cortex, a brain structure where neither APP nor APP-LGF mice showed any alteration in CD36 protein levels (Figure 5C).
