Figure 2.4: FBXO7-/- mouse brains display no α-synuclein- or APP-positive inclusions: (a) Representative images from 5 μm thick sagittal paraffin-embedded brain sections of the cortical region of P18 FBXO7+/+ and FBXO7-/- mice. Whole brain sections were subjected to immunohistochemistry using the α-synuclein antibody and screened for inclusions. Scale bar equals 40 μm. (b) Cortical lysates of P18 FBXO7+/+ and FBXO7-/- mice were subjected to immunoblotting using the α-synuclein and γ-tubulin antibodies. The latter served as a loading control. (c) Densitrometric quantification of α-synuclein protein levels in (b). n= 5 FBXO7 litterpairs were analyzed (Paired t-test, ns= non significant, mean + s.e.m.). (d) Representative images from 5 μm thick sagittal paraffin-embedded brain sections of the corpus callosum and cerebellar white matter region of P18 FBXO7+/+ and FBXO7-/- mice. Whole brain sections were subjected to immunohistochemistry using the APP antibody and screened for inclusions. Scale bar equals 40 μm. (e) Cortical lysates of P18 FBXO7 +/+ and FBXO7 -/- mice were subjected to immunoblotting using the APP and γ-tubulin antibodies. The latter served as a loading control. (f) Densitrometric quantification of APP protein levels in (e). n= 8 FBXO7 litterpairs were analyzed (Paired t-test, ns= non significant, mean + s.e.m.).
2.4 Increased astrogliosis and elevated levels of apoptosis in the brain of FBXO7-/- mice
Increased inflammation is associated with several neurodegenerative diseases, including Parkinson's disease (Damier et al., 1993; Dickson, 2012). I hence compared sagittal brain sections of P18 FBXO7+/+ and FBXO7-/- mice first for astrogliosis using the glial fibrillary acidic protein (GFAP) antibody. Indeed, GFAP levels were increased in the FBXO7-/- animals (Figure 2.5 a, b). I also examined for increased microgliosis using the Iba1 antibody on the same brain sections but found no difference (Figure 2.5 c, d, e).
2.4 Increased astrogliosis and elevated levels of apoptosis in the brain of FBXO7-/- mice
2.4 Increased astrogliosis and elevated levels of apoptosis in the brain of FBXO7-/- mice
Figure 2.5: FBXO7-/- mice show increased levels of astrogliosis in the brain: (a) Representative images from 5 μm thick sagittal paraffin-embedded brain sections of the cortical region of P18 FBXO7+/+ and FBXO7-/- mice. Whole brain sections were subjected to immunohistochemistry using the GFAP antibody. Scale bar equals 40 μm. (b) Quantification of GFAP-positive area (% stained area/mm²) in the cortex of at least three independent FBXO7+/+ and FBXO7-/- littermates. Three anatomically matched sections per animal were quantified in a blinded manner and analyzed using a custom-designed macro. (Paired t-test, *p<0.05, mean + s.e.m.). (c) Representative images from 5 μm thick sagittal paraffin-embedded brain sections of the cortical region of P18 FBXO7 +/+ and FBXO7 -/- mice. Whole brain sections were subjected to immunohistochemistry using the Iba1 antibody. Scale bar equals 40 μm. (d) Quantification of Iba1-positive area (% stained area/mm²) in the cortex of at least three independent FBXO7 +/+ and FBXO7 -/- littermates. Three anatomically matched sections per animal were quantified in a blinded manner and analyzed using a custom-designed macro. (Paired t-test, ns= non significant, mean + s.e.m.). (e) Quantification of Iba1-positive cells (cells/mm²) of the same brain sections quantified in (d). Quantification was done manually and in a blinded manner. (Paired t-test, ns= non significant, mean + s.e.m.).
Cell death and apoptosis are major factors in neurodegenerative diseases and are usually accompanied by inflammation (Wyss-Coray and Mucke, 2002). I hence determined, whether brain sections of P18 FBXO7+/+ and FBXO7-/- mice showed any abnomality in cell death levels using a commercial TUNEL kit. The TUNEL kit detects sites of fragmented DNA that result from apoptotic pathways. Interestingly, many of the brain regions analyzed (cortex, cerebellum, hippocampus) showed a tendency to elevated levels of apoptotic cells being only in the cortex significantly increased (Figure 2.6 a, b). Levels of apoptotic cells in the mibrain (Figure 2.6 c, d) as well as the number of dopaminergic in the substantia nigra were unchanged (Data Siv Vingill).
2.4 Increased astrogliosis and elevated levels of apoptosis in the brain of FBXO7-/- mice
2.4 Increased astrogliosis and elevated levels of apoptosis in the brain of FBXO7-/- mice
Figure 2.6: FBXO7-/- mice display increased apoptosis in the brain: (a) Representative images from 5 μm thick sagittal paraffin-embedded brain sections of the cortical, cerebellar and hippocampal region of P18 FBXO7+/+ and FBXO7-/- mice. Whole brain sections were subjected to immunohistochemistry using a TUNEL detection kit, staining apoptotic cells. Arrowheads indicate apoptotic cell bodies. Scale bar equals 40 μm. (b) Quantification of TUNEL-positive cells (cells/mm²) in the cortex, cerebellum and hippocampus. Three anatomically matched sections per animal were quantified manually in a blinded manner. (Paired t-test,
*p<0.05, ns= non significant, mean + s.e.m.). (c) Representative images from 5 μm thick coronal paraffin-embedded brain sections of the midbrain region of P18 FBXO7+/+ and FBXO7-/- mice. Whole brain sections were subjected to immunohistochemistry using a TUNEL detection kit, staining apoptotic cells. Arrowheads indicate apoptotic cell bodies. Scale bar equals 40 μm. (d) Quantification of TUNEL-positive cells (cells/mm²) in the midbrain. Three anatomically matched sections per animal were quantified manually in a blinded manner. (Paired t-test, ns= non significant, mean + s.e.m.).
To corroborate the increased levels of apoptosis observed in vivo, I performed a survival assay by acutely knocking down FBXO7 in cultured cortical neurons. For this, I transfected cortical neurons at day in vitro (DIV) 3 with plasmids encoding either the control vector, functional FBXO7 shRNA or non-functional shRNA (Figure 2.7 b) and quantified the number of pyknotic nuclei and level of cleaved caspase 3 at DIV 7. The number of apoptotic neurons was increased by more than 2.5 fold in the condition, in which FBXO7 was knocked down as compared to control cells (Figure 2.7 a), suggesting that FBXO7 is essential for cortical neuron survival both in vivo and in vitro.
Figure 2.7: FBXO7 is required for neuronal survival: (a) Apoptotic cortical neurons were counted manually in a blind manner and quantified. At least four independent experiments were included in the analysis (ANOVA, **p<0.01, mean + s.e.m.). (b) Lysates from cultured rat cortical neurons were nucleofected at DIV0 with either control pSuper, functional FBXO7 shRNA or non-functional FBXO7 as an additional control.
Neurons were harvested four days after nucleofection and subjected to immunoblotting with the FBXO7 and 14-3-3 antibodies. The latter served as a loading control.