L-Lactate Promotes Adult Hippocampal Neurogenesis

Neurogenesis, the formation of new neurons in the adult brain, is important for memory formation and extinction. One of the most studied external interventions that affect the rate of adult neurogenesis is physical exercise. Physical exercise promotes adult neurogenesis via several factors including brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). Here, we identified L-lactate, a physical exercise-induced metabolite, as a factor that promotes adult hippocampal neurogenesis. While prolonged exposure to L-lactate promoted neurogenesis, no beneficial effect was exerted on cognitive learning and memory. Systemic pharmacological blocking of monocarboxylate transporter 2 (MCT2), which transports L-lactate to the brain, prevented lactate-induced neurogenesis, while 3,5-dihydroxybenzoic acid (3,5-DHBA), an agonist for the lactate-receptor hydroxycarboxylic acid receptor 1 (HCAR1), did not affect adult neurogenesis. These data suggest that L-lactate partially mediates the effect of physical exercise on adult neurogenesis, but not cognition, in a MCT2-dependent manner

Sirt6 alters adult hippocampal neurogenesis

<div><p>Sirtuins are pleiotropic NAD⁺ dependent histone deacetylases involved in metabolism, DNA damage repair, inflammation and stress resistance. SIRT6, a member of the sirtuin family, regulates the process of normal aging and increases the lifespan of male mice over-expressing Sirt6 by 15%. Neurogenesis, the formation of new neurons within the hippocampus of adult mammals, involves several complex stages including stem cell proliferation, differentiation, migration and network integration. During aging, the number of newly generated neurons continuously declines, and this is correlated with a decline in neuronal plasticity and cognitive behavior. In this study we investigated the involvement of SIRT6 in adult hippocampal neurogenesis. Mice over-expressing Sirt6 exhibit increased numbers of young neurons and decreased numbers of mature neurons, without affecting glial differentiation. This implies of an involvement of SIRT6 in neuronal differentiation and maturation within the hippocampus. This work adds to the expanding body of knowledge on the regulatory mechanisms underlying adult hippocampal neurogenesis, and describes novel roles for SIRT6 as a regulator of cell fate during adult hippocampal neurogenesis.</p></div

Sirt6-OE does not affect total numbers of DG and cortical NeuN⁺ or hippocampal and cortical S100β⁺ cells.

<p><b>(A)</b> Left panel: Total numbers of NeuN⁺ cells were counted in the DG of WT (n = 6) and Sirt6-OE (n = 6) mice. Right panel: representative NeuN⁺ cells in the DG. Scale bar = 100μm <b>(B)</b> DG distribution of NeuN⁺ cells throughout the DG <b>(C)</b> Density of NeuN⁺ cells in the DG. (<b>D</b>) Density of NeuN⁺ cells in the cortex. <b>(E)</b> Left panel: Total numbers of S100β⁺ cells were counted in the hippocampi of WT (n = 6) and Sirt6-OE (n = 6) mice. Right panel: arrow indicates representative S100β⁺ cells in the hippocampus. Scale bar = 100μm <b>(F)</b> Hippocampal distribution of S100β⁺ cells. <b>(G)</b> Density of S100β⁺ cells in the hippocampus. () Density of S100β⁺ cells in the in the lateral parietal association cortex (LPtA) and primary somatosensory trunk cortex (S1Tr).</p

Sirt6-OE alters hippocampal neurogenesis.

<p><b>(A)</b> Sox2⁺/BrdU⁺ cells were counted for each animal's SGZ in random bregma positions using the optical fractionator probe (Stereoinvestigator software, MBF bioscience), and total SGZ population estimation was extrapolated. Results were averaged across genotype group. No significant difference was found in total Sox2⁺/BrdU⁺ cells (t-test, p = 0.71). <b>(B)</b> Left panel: Distribution of Sox2⁺/BrdU⁺ neural progenitor cells in the SGZ of WT and Sirt6-OE mice. Results were averaged across genotype groups for each hippocampal slice according to distance from bregma position. No significant effect was found for genotype (2-way ANOVA, P = 0.14). Right panel: representative Sox2⁺/BrdU⁺ cell in the SGZ of WT and SIRT6-OE mice. <b>(C)</b> DCX⁺/BrdU⁺ cells were counted in the granular layer at random distances from bregma using an optical fractionator probe, and total SGZ population estimation was extrapolated for each animal. Results were averaged each across genotype. A higher, but not significant, number of total DCX⁺/BrdU⁺ cells were observed in Sirt6-OE mice compared to WT littermates (3022 ± 254.7 and 2469 ± 170.1 for WT and Sirt6-OE mice respectively, P = 0.1069, t-test). <b>(D)</b> Left panel: Distribution of DCX⁺/BrdU⁺ neural progenitor cells in the SGZ of WT and Sirt6-OE mice. Results were averaged across genotype group for each hippocampal slice according to distance from bregma. A significant genotype effect was found (2-way ANOVA, P = 0.005). Right panel: representative DCX⁺/BrdU⁺ cell in the SGZ of WT and SIRT6-OE mice. <b>(E)</b> NeuN⁺/BrdU⁺ cells were counted in the granular layer in random distances from bregma using an optical fractionator probe, and total SGZ population estimation was extrapolated for each animal. Results were averaged across genotype group. A lower, but not significant, number of total NeuN⁺/BrdU⁺ cells were observed in Sirt6-OE mice compared to WT littermates (1228 ± 90.68 and 1076 ± 95.36 for WT and Sirt6-OE mice respectively, P = 0.26, t-test). <b>(F)</b> Left panel: Number of total NeuN⁺/BrdU⁺ mature neurons in the SGZ of WT and Sirt6-OE mice. NeuN⁺/BrdU⁺ cells were counted in the granular layer in random distances from bregma in each animal using an optical fractionator probe. Results were averaged across genotype group for each hippocampal slice according to distance from bregma. A significant genotype effect was found (two-way ANOVA, F_(1,19) = 3.989, p = 0.018 for genotype effect). Right panel: representative NeuN⁺/BrdU⁺ cell in the SGZ of WT and SIRT6-OE mice. <b>(G)</b> Protein levels of WT and Sirt6-OE mice. Western blot analysis of WT and Sirt6-OE mice, blotted for SIRT6 and the house-keeping protein, α-tubulin. A robust increase in SIRT6 levels is clearly seen in Sirt6-OE mice. <b>()</b> Hippocampal SIRT6 protein levels from mice with high and low early neuron population numbers in the Sirt6-OE group. Upper panel. Western blot analysis of SIRT6 levels in Sirt6-OE mice with the highest and lowest new early neuron population counts, compared to a WT mouse with an average population count. Lower panel: Densitometric quantification of the corresponding bands was performed using ImageJ analysis software and each group was averaged. <b>(I)</b> NeuN⁺/BrdU⁺ cells were counted in the granular layer in random distances from bregma in each animal using an optical fractionator probe. Results were averaged across genotype for each bregma position and divided by the DG volume. A significant genotype effect was found (two-way ANOVA, F_(1,19) = 10.09, * p = 0.0017 for genotype main effect). Immunofluorescence images were taken at 10x magnification, scale bar = 20μm. Insets represent 63x magnification.</p

Experimental design.

<p>Experimental design.</p

Sirt6-OE does not affect hippocampal distribution of astrocytes.

<p>Bivariate distribution of astrocytes scattering in the hippocampi of SIRT6 OE and WT mice. (A) Two dimensional scattering distributions of astrocytes in the following distances from bregma: -1.22, -1.34, -1.82, -1.94, -2.18, -2.46, -2.7, -2.92, -3.08, -3.16 of SIRT6 OE (<i>n</i> = 6) and WT (<i>n</i> = 6) hippocampi, logarithmic scale of cell count per square bin (<i>bin size</i> = 18² μm). <b>(B)</b> Similarity of astrocyte distributions to a hypothetical uniform distribution of the same size and shape in the two-dimensional space as a function of bregma, expressed as the confidence level (1—P value), two-sample Kolmogorov-Smirnov test.</p

Cortical thickness of WT and Sirt6-OE mice.

<p><b>(A)</b> Cortical thickness was measured perpendicular to the CA1 in the same location for each bregma investigated and averaged across genotype group. Measurements were made using Stereo investigator software (MBF bioscience). No significant difference was seen between the two groups (2-way ANOVA, p = 0.35). <b>(B)</b> Hippocampal area along the brain of WT and Sirt6-OE mice. Hippocampal area was measured by contouring the hippocampal slices for each bregma using Stereo investigator software (MBF bioscience) and averaging its surface area across genotype group. No significant difference was seen between the two groups (2-way ANOVA, P = 0.19). <b>(C)</b> Area of the dentate gyrus along the hippocampus of WT and Sirt6-OE mice. Dentate gyrus area was measured by contouring the DG region for each bregma using Stereo investigator software (MBF bioscience) and averaging its surface area across genotype group. No significant difference was seen between the two groups (2-way ANOVA, p = 0.48).</p

Evidence for a developmental role for TLR4 in learning and memory

Toll-like receptors (TLRs) play essential roles in innate immunity and increasing evidence indicates that these receptors are expressed in neurons, astrocytes and microglia in the brain where they mediate responses to infection, stress and injury. Very little is known about the roles of TLRs in cognition. To test the hypothesis that TLR4 has a role in hippocampus-dependent spatial learning and memory, we used mice deficient for TLR4 and mice receiving chronic TLR4 antagonist infusion to the lateral ventricles in the brain. We found that developmental TLR4 deficiency enhances spatial reference memory acquisition and memory retention, impairs contextual fear-learning and enhances motor functions, traits that were correlated with CREB up-regulation in the hippocampus. TLR4 antagonist infusion into the cerebral ventricles of adult mice did not affect cognitive behavior, but instead affected anxiety responses. Our findings indicate a developmental role for TLR4 in shaping spatial reference memory, and fear learning and memory. Moreover, we show that central TLR4 inhibition using a TLR4 antagonist has no discernible physiological role in regulating spatial and contextual hippocampus-dependent cognitive behavior

Developmental TLR4 deficiency, but not pharmacological TLR4 antagonism, enhances retention of spatial reference memory.

<p>(<b>A</b>) TLR4^−/− (n = 19) and TLR4^+/+ (n = 24) mice were tested in probe trials at 24, 48, 72 and 96 hours following training for retention of spatial reference memory. Tests were done after all experimental groups exhibited loss of memory of the platform location. Mean distance from the platform was measured and used to indicate efficiency in locating the hidden platform. TLR4^−/− mice showed shorter mean distance from the platform at 24 and 48 hours after training compared with TLR4^+/+ mice, indicating a more accurate swim toward the platform quadrant (<b>B</b>) Mice (C57BL/6) were implanted with an osmotic pump containing either aCSF (n = 10) or a TLR4 antagonist (n = 10). The pump was connected via tubing to a cannula, which was positioned to the lateral ventricle. Following training in the MWM task, mice were tested in probe trials at 24 and 48 hours following training for retention of spatial reference memory. Both experimental groups exhibited similar performance during probe trials, as measured by mean distance from the platform.</p

CREB and p-CREB are upregulated in the hippocampus of TLR4^−/− mice.

<p>Brains from TLR4^+/+ (n = 8) and TLR4^−/− (n = 8) mice were dissected and hippocampi were removed. Tissues were then lysed, electrophoresed and immunoblotted against GluR1, CREB, ERK and their phosphorylated forms. Representative blots demonstrate that levels of CREB and pCREB were upregulated in TLR4^−/− mice compared to TLR4^+/+ mice, whereas GluR1, ERK and their phosphorylated forms were not changed. * p<0.05.</p