Slowing Alzheimer's disease progression through probiotic supplementation

Abstract

The lack of affordable and effective therapeutics against cognitive impairment has promoted research toward alternative approaches to the treatment of neurodegeneration. In recent years, a bidirectional pathway that allows the gut to communicate with the central nervous system has been recognized as the gut-brain axis. Alterations in the gut microbiota, a dynamic population of trillions of microorganisms residing in the gastrointestinal tract, have been implicated in a variety of pathological states, including neurodegenerative disorders such as Alzheimer's disease (AD). However, probiotic treatment as an affordable and accessible adjuvant therapy for the correction of dysbiosis in AD has not been thoroughly explored. Here, we sought to correct the dysbiosis in an AD mouse model with probiotic supplementation, with the intent of exploring its effects on disease progression. Transgenic 3xTg-AD mice were fed a control or a probiotic diet (Lactobacillus plantarum KY1032 and Lactobacillus curvatus HY7601) for 12 weeks, with the latter leading to a significant increase in the relative abundance of Bacteroidetes. Cognitive functions were evaluated via Barnes Maze trials and improvements in memory performance were detected in probiotic-fed AD mice. Neural tissue analysis of the entorhinal cortex and hippocampus of 10-month-old 3xTg-AD mice demonstrated that astrocytic and microglial densities were reduced in AD mice supplemented with a probiotic diet, with changes more pronounced in probiotic-fed female mice. In addition, elevated numbers of neurons in the hippocampus of probiotic-fed 3xTg-AD mice suggested neuroprotection induced by probiotic supplementation. Our results suggest that probiotic supplementation could be effective in delaying or mitigating early stages of neurodegeneration in the 3xTg-AD animal model. It is vital to explore new possibilities for palliative care for neurodegeneration, and probiotic supplementation could provide an inexpensive and easily implemented adjuvant clinical treatment for AD.

Keywords: Alzheimer’s disease; entorhinal cortex; glia; gut microbiota; hippocampus; inflammation; neurodegeneration; neurons.

Figure 1

Spatial orientation and memory improved in 3xTg mice treated with probiotics. Each data point represents an individual mouse from a single trial. Each group is represented by 4 weeks of trials for each animal per group (n = 5) for escape latency and head pokes, respectively. (A) Overall escape latency times represent average escape latency for each group ± SD across all maze trials. Data reported in seconds, as average latency for each group. (B) Number of head pokes represent the average total number of erroneous attempts for each group ± SD across all maze trials. Statistically significant differences among groups were analyzed via one-way ANOVA at a 95% confidence interval, using Tukey’s multiple comparisons post hoc test. **p < 0.01, ****p < 0.0001.

Figure 2

No changes in cortical neuronal populations after treatment with probiotics. (A) Representative immunofluorescent staining in layers 4/5 of the entorhinal cortex at 7 months of age, using a NeuN⁺ antibody to tag neurons (top panels) and DAPI nuclear stain to detect total cells (middle panels). Merged images shown in bottom panels. (B) In layers 4/5 of the entorhinal cortex, there is no significant difference between the relative number of neurons (NeuN⁺/DAPI⁺) in WTC, ADC and ADP treated mice. Each data point represents the average (n = 3 fields per section) percentage of neurons out of total cells per section stained in each group of 5 animals. Data reported as mean percentage for each group ± SEM and analyzed by a Kruskal–Wallis test.

Figure 3

Probiotic treatment affected cortical astrocyte populations in AD mice. (A) Representative immunofluorescent staining in layers 4/5 of the entorhinal cortex at 10 months of age using a GFAP antibody to tag astrocytes (top panels) and DAPI nuclear stain to detect total cells (middle panels). Merged images shown in bottom panels. (B) Higher percentage of astrocytes were present in the entorhinal cortex of ADC mice compared to WTC, while ADP % astrocytes were comparable to WTC mice. Each data point represents the average (n = 3 fields per section) percentage of GFAP out of total cells per section stained in each group of 5 animals. Data reported as mean percentage for each group ± SEM and analyzed by a Kruskal–Wallis test.

Figure 4

Probiotic treatment altered only cortical astrocytes in AD mice. Each data point represents the average (n = 3 fields per section) percentage of neurons or astrocytes out of total cells counted per section stained. For each mouse (n = 5 per group), 2–3 sections were stained and pooled per group (n = 10–12). (A) No changes in cortical neurons (NeuN⁺) were observed in ADC versus ADP, while (B) a significant decrease in astrocytes (GFAP⁺) was observed in the entorhinal cortex of 10 months old ADP mice on a 12-week probiotic diet compared to ADC mice. Data reported as mean percentage per group ± SEM and analyzed by a Mann–Whitney test. *p < 0.05.

Figure 5

Probiotics treatment protected neuronal cells in the anterior hippocampus in AD mice. Each data point represents the average (n = 3 fields per section) percentage of positive cells out of total cells per section stained in each group. For each group, 5 mice were assessed. Based on Bregma location, results were subdivided into anterior vs. posterior area (n = 6–10 per area). NeuN⁺, GFAP⁺ and Iba1⁺ cells were counted in the (A) anterior and (B) posterior hippocampi of ADC and ADP mice. Significant differences were observed in neurons but not glia in anterior hippocampi. Data reported as mean percentage per group ± SEM and analyzed by Mann–Whitney test. **p < 0.01.

Figure 6

Neural cell populations in female mice were significantly affected by probiotics treatment in the anterior hippocampus. Percentages of (A) neurons (NeuN⁺), (B) astrocytes (GFAP⁺) and (C) microglia (Iba1⁺) were evaluated in 10 months old AD female mice. Given the small number of females in each group (n = 3 ADP and n = 4 ADC), each data point represents the average counts of 3 fields per section. All stained sections of each animal in a group are represented in the group. Data reported as mean percentage per group and analyzed by Mann–Whitney test. *p < 0.05.

Figure 7

Relative abundance boxplots of Bacteroidetes and Firmicutes. Phylum-level relative abundances are plotted by group with baseline (red) indicating samples from all 10 mice before treatment (week 0), control (green) indicating the 5 mice that did not receive a probiotic supplement (week 12), and the probiotic (blue) group that received the probiotic treatment (week 12). A significant difference was observed when comparing the baseline samples to the probiotic samples for Bacteroidetes abundance (p < 0.05). Boxplots are presented in Tukey-style. The minimum sequencing depth for these samples was 14,112 reads.