**HD—mouse model studies**

There are several commercially available mouse models of HD. They differ in the genetic background, the structure of the transgene, and the disease phenotype. The most commonly used lines are R6/1 and R6/2, which are characterized by early symptoms and rapid progression of the disease, compared to the BACHD line. The BACHD mouse model shows the first symptoms of the disease between 2 and 6 months of age, but their severity appears after about a year. The BACHD line shows somatic stability in embryos [86].

Studying the microbiome is an increasingly emerging trend in HD research. One study showed an impact of the transplantation of a microbiome derived from WT mice into a mouse model of HD on its phenotype. The results show that especially the females responded positively to this procedure, as improvements in cognitive function have been observed in animals suffering from HD. The same study proved the ineffectiveness of this approach in males. Researchers speculated that the possible reasons for that phenomenon

might be more extensive changes in structure, instability in the gut microbiome and the imbalance in acetate immune profiles [87]. In order to characterize the gut microbiome in a mouse model of HD, 16S RNA sequencing was performed. The research was carried out on R6/1 mice. Sequencing results revealed significant differences in the composition of the microbiome. Furthermore, the amount of water in the feces of HD mice at 12 weeks of age was significantly changed. Most notably, there was an increase in *Bacteroidetes* and a proportional decrease in *Firmicutes*. Interestingly, an increase in microbiome diversity was also observed in HD males compared to WT control mice, but these differences were not observed in females. The changes coincided with an increased food intake and a simultaneous decrease in body weight [88]. It has been proven that PD is characterized by a decrease in the expression of TJ proteins, which under physiological conditions maintain the integrity of the intestinal barrier [89]. Björkqvist and coauthors evaluated whether the same mechanism is responsible for the pathologies occurring in another mouse model of HD (R6/2). The results showed a significant decrease in body weight and body length in these mice. They were also accompanied by a decrease in colon length compared to WT mice, but TJ protein levels showed no statistically significant changes between groups. Moreover, along with the observed changes, differences in the composition of the gut microbiota were also found in the R6/2 mice. Increased amounts of *Bacteroidetes* and *Proteobacteria* and decreased amounts of *Firmicutes,* relative to levels maintained in the control group were demonstrated [90]. A very interesting and detailed study was performed by Gubert et al. They focused on comparing the study group (R6/1 mouse line), which consisted of 3 subgroups: animals with standard living conditions, mice with additional environmental enrichment, and groups of animals with increased physical activity, with WT mice as controls. The results indicated a possible modulation of the gut microbiome by the environment. Therapeutic effects on psychomotor symptoms and the brain have been reported in groups of animals with an enriched environment and greater activity compared to the control group. Changes in the composition of the microbiome at the level of orders such as *Bacteroidales*, *Lachnospirales,* and *Oscillospirales* have also been demonstrated. The results obtained in this experiment show higher alpha diversity for all HD mice compared to WT mice. There was no difference in food intake, but there was a previously expected decrease in body weight in the HD mice compared to the control group. Increased water intake by animals from the test groups was shown, which was associated with the increase in alpha diversity. With the aging of the HD animals, increased fecal excretion was noted. Post-mortem analysis showed a statistically significant decrease in the brain weight of HD mice. There were also significant differences between males and females. The brain weight of females was lower in the group of mice with standard living conditions. Based on the study of the concentration of SCFAs and branched chain fatty acids (BCFAs) in the feces, an attempt was made to check what role these metabolites may play in living condition changes. Male mice from the group with increased physical activity were characterized by a decrease in the concentration of butyrate and valerate. There was no correlation between the concentration of substances, such as acetate and propionate, and the living conditions, genotype, or gender. Statistically significant differences were found between HD and WT mice in the alpha diversity index. The test groups showed increased alpha diversity indices in contrast to the control group. The results of the beta diversity analysis showed differences between the sexes of the animals. Certain orders of microbial bacteria have been identified as those that play the greatest role in microbiome changes under different animal housing conditions. These include the orders *Bacteroidales*, *Lachnospirales,* and *Oscillospirales* [91].

Early pathological features associated with HD are molecular deficits in myelination and progressive neurodegeneration. Experiments conducted on germ-free (GF) animals suggested that there is a two-way communication between the microbiome, gut, and brain [11,92]. Research conducted on the BACHD mouse model was intended to answer the question of what impact the microbiome has on myelin plasticity and oligodendrocyte dynamics. The experiment compared GF, specific pathogen-free (SPF), and WT mice. Ani-

mals of both sexes were used in the experiment. Analysis of myelin in the corpus callosum revealed changes in myelin thickness in BACHD GF mice compared to SPF mice, while no intergroup changes were observed in WT mice. However, significant differences in myelin density were noted in all groups compared to WT SPF mice. In the GF conditions, a reduced level of myelin-associated proteins, such as myelin basic protein (MBP), proteolipid protein (PLP), and Ermin (Ermn), and a lower number of mature oligodendrocytes in the prefrontal cortex were observed compared to the SPF conditions, regardless of the mouse genotype. Slight differences in family and genus were also observed in the commensal bacteria of the gut microbiome in the BACHD and WT groups maintained under SPF conditions. However, the differences were not statistically significant. Researchers concluded that the *HTT* mutation in BACHD mice does not cause profound disturbances in the intestinal microflora, and thus plasticity defects are not associated with disturbances in the structure of the microbiome. Analysis of the brain structures of GF animals showed that then environment had a greater effect on the myelination caliber of callosum axons in BACHD animals compared to WT controls, while a possible distribution of myelin plaques was observed in both genotypes. The axons of mice maintained under GF conditions were characterized by a reduced diameter and a lower g-ratios, which could suggest thicker myelin. Examination of the myelin membranes, however, showed that the observed features may have been due to the decompaction of the laminae and not an increase in their number. A similar trend of increased periodicity, suggesting decompaction, was also observed in BACHD mice under SPF conditions compared to WT, prompting the conclusion that the *HTT* mutation in BACHD animals causes this pathology. Supportive is the observation of a trend towards lower levels of the cortical myelin-associated proteins MBP and PLP, which play a key role in myelin compaction. The researchers did not observe significant changes in the gut bacterial community. Slight disparities were observed in BACHD mice at 3 and 6 months of age compared to WT mice, with reduced numbers of *Prevotella* and *Bacteroides* at the genus level and part of the *Bacteroidetes* type [93]. More reports indicate the importance of the intestinal microbiome in the communication between the digestive system and the brain and its impact on the pathologies of neurodegenerative diseases. Subsequent studies involved shotgun sequencing of the gut microbiome from R6/1 mice, aged 4–12 weeks (from early adolescent to adult stages). Metabolomic analyses, in addition to those performed on fecal samples, were also performed on blood plasma collected from 12-week-old animals. The results showed an upregulation of bacterial gene expression, which may indicate potential early effects of the HTT protein mutation in the gut. In addition, mice at 12 weeks of age were found to have disturbed gut microbiome function. In particular, the researchers' attention was drawn to the increase in the butanoate metabolic pathway, which leads to increased production of SCFA playing a protective role. This increase was not observed when analyzing plasma from 12-week-old mice. Statistical analysis of the results obtained in metagenomic and metabolomic studies allowed for the observation of a negative correlation of several species of *Bacteroides* with ATP and pipecolic acid in plasma. During the experiment, feces were collected at five different time points. No statistically significant differences in the composition of the microbiome were observed when comparing the mice from the study group and the control WT group. The dominance of two phyla, *Bacteroidetes* and *Firmicutes,* was observed, followed by the *Proteobacteria*, *Actinobacteria,* and *Verrucomicrobia* phyla. It was determined that at the family level, the most numerous group was *Lachnospiraceae*, followed by similar numbers in the groups of *Bacteroidaceae*, *Porphyromonadaceae*, *Prevotellaceae,* and *Clostridiaceae*. No statistically significant differences were found between bacterial families at any timepoint when comparing WT mice. At 12 weeks of age, which corresponds to the timepoint before the onset of overt motor symptoms in HD mice, differences in 30 bacterial species were observed between HD and WT mice. These included *Clostridium mt 5*, *Treponema phagedenis*, *Clostridium leptum CAG: 27*, *Desulfatirhabdium butyrativorans*, *Plasmodium chabaudi*, *Defulfuribacillus alkaliarsenatis*, *Plasmodium yoelii,* and *Chlamydia abortus*. No differences in the abundance of butyrate

producers such as *Roseburia intestinalis*, *Clostridium symbiosum,* and *Eubacterium rectale* were found when comparing samples from HD and WT mice [94].
