A ketogenic diet slows down excessive weight loss in Hexa-/-Neu3-/- mice
Diet calorie composition is derived mainly from fat for the ketogenic diet. However, calorie composition is derived chiefly from carbohydrates for the standard diet (Fig. 2A). Tay-Sachs disease mouse model body weight significantly decreased progressively until death, but ketogenic diet intervention slowed excessive weight loss (Fig. 2B). KD and KD + PG mice groups consumed similar quantities of the ketogenic diet (KD: 2,72 ± 0,67 g/day; KD + PG: 2,55 ± 0,26 g/day) for ten days (Fig. 2C). Hexa-/-Neu3-/- mice which are KD (n = 9) and KD + PG (n = 9) treatment groups, displayed elevation of blood BHB levels compared to SD-intervened Hexa-/-Neu3-/- mice from 0,45 ± 0,129 mM to 1,4 ± 0,683 mM and 0,45 ± 0,129 mM to 1,475 ± 0,33 mM respectively (Fig. 2D).
Combined treatment significantly reduces expression levels of chemokines in the cortex.
Neuroinflammation triggers the secretion of pro-inflammatory cytokines/chemokines from macrophages in astrocyte and microglial cells. The result showed that 140-day-old Hexa-/-Neu3-/- mice displayed elevation of pro-inflammatory cytokine/chemokines gene expressions in the cortex. It has been demonstrated that Ccl2 or Mcp-1(monocyte chemoattractant protein-1) is a cytokine involved in regulating immune cell infiltration and blood-brain barrier integrity (Cazareth et al., 2014; Yao & Tsirka., 2014). At the same time, Ccl3 plays an influential role in the movement of lymphocytes, monocytes, and neutrophils with Ccl2 (Reichel et al., 2009). Ccl5 or RANTES also assists the chemotaxis of T-cells and monocytes by interacting with various receptors (Murooka et al., 2008). In addition, Cxcl10 is another pro-inflammatory chemokine secreted by astrocytes that facilitates T-lymphocyte migration to tissue damage (Klein et al., 2005). Overall, each treatment which was the KD, PG, and combination of the KD and PG, significantly reduced gene expression levels of pro-inflammatory cytokines and chemokine (Ccl2, Ccl3, Ccl5, Cxcl10) and astrocyte markers (GFAP) in the cortex of Hexa-/-Neu3-/- mice (Fig. 3A-E). In addition to the neuroinflammatory role of chemokines, they also play essential roles in regulating neuronal death (Ramesh et al., 2013). For example, the Ccl2-knockout mouse model protects the neuron from TD (thiamine deficiency)-induced cell death due to macrophage/microglial activation (Yang et al., 2011). Moreover, Cxcl10 overexpression has been detected in neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease (Zaheer et al., 2013; Kalkonde et al., 2007). Ketogenic diet treatment but no other treatments increased expression level of anti-apoptotic gene expressions. Interestingly, not only Bax, one of the pro-apoptotic genes, expression level increased after ketogenic diet treatment, but also Bcl-2 and Bcl-xL, which are anti-apoptotic genes, expression levels increased (Fig. S1).
The combined treatment alleviates astrogliosis in Hexa-/-Neu3-/- mice.
Glia or neuroglia are the non-neuronal cells that mainly include astrocytes and microglia in the central nervous system. Microglial cells are resident immune cells with phagocytic activity to protect the central nervous system. Once activated, microglia move to damaged tissue and secrete pro-inflammatory cytokine/chemokine to scavenge dead cells by phagocytosis (Colonna & Butovsky., 2017). In addition, active microglia trigger the macrophages/monocyte's action and activation of astrocytes (Liddelow et al., 2017).
It has been shown that the accumulation of undegraded macromolecules triggers a neuroinflammatory response in various LSDs (Vitner et al., 2010). Akyıldız-Demir et al. previously reported that GM2 ganglioside accumulation induces neuroinflammation in the TSD mice brain cortex. Here we showed that KD and PG treatments did not alter the GM2 ganglioside level shown with thin-layer chromatography in the cortex (Fig S2); however, the gene expression level of enzymes involved in ganglioside biosynthesis (B4Galnt1, B3Galnt4, GM3S, GD3S, and HexB) was altered only with KD treated mice (Fig S3). To visualize the effects of a combined ketogenic diet and anti-inflammatory drug treatment against neuroinflammation, 140-day-old Hexa-/- and Hexa-/-Neu3-/- mice brain sections were stained with anti-MOMA-2 and anti-GFAP which are specific for macrophage/monocyte and astrocyte respectively. MOMA-2-positive cells significantly increased in 140-day-old Hexa-/-Neu3-/- mice brain cortex compared to age-matched Hexa-/- mice (Fig. 4A-C). MOMA-2-positive cells were significantly reduced solely after KD treatment but not after anti-inflammatory drugs and combining ketogenic diet and anti-inflammatory drug treatments. In contrast to MOMA-2 staining results, the GFAP-positive cells were approximately halved after the ketogenic diet, anti-inflammatory, and a combination of the ketogenic diet and anti-inflammatory drug treatments (Fig. 4B-D). In this study, KD treatment improved both astrogliosis and macrophage activity. However, a combined KD and PG did not significantly affect macrophage/monocyte activity in the 140-day-old Tay-Sachs mouse brain cortex.
Treatment with the ketogenic diet and propagermanium provides no improvement in anxiety-related behavior and neuromotor activities.
The open-field test was used to measure levels of anxiety-related behavior via mice’s body activity and locomotion. Combined ketogenic diet and propagermanium did not alter time spent in the periphery and center of the platform, and total distance traveled for 140-day-old Hexa-/-Neu3-/- mice compared to age-matched Hexa-/- (Fig. 5A).
140-day-old Hexa-/-Neu3-/- mice displayed a progressive reduction of neuromotor activity in the rotarod test that was previously published. After each treatment strategy, 140-day-old Hexa-/-Neu3-/- mice did not stay enough on the accelerated rod compared to age-matched Hexa-/- mice. This indicated that each treatment strategy did not improve motor coordination and balance in Hexa-/-Neu3-/- mice (Fig. 5B).