Folic Acid and Vitamin B12 Prevent Deleterious Effects of Rotenone on Object Novelty Recognition Memory and Kynu Expression in an Animal Model of Parkinson's Disease

Abstract

Parkinson's disease (PD) is characterized by a range of motor signs, but cognitive dysfunction is also observed. Supplementation with folic acid and vitamin B12 is expected to prevent cognitive impairment. To test this in PD, we promoted a lesion within the substantia nigra pars compacta of rats using the neurotoxin rotenone. In the sequence, the animals were supplemented with folic acid and vitamin B12 for 14 consecutive days and subjected to the object recognition test. We observed an impairment in object recognition memory after rotenone administration, which was prevented by supplementation (p < 0.01). Supplementation may adjust gene expression through efficient DNA methylation. To verify this, we measured the expression and methylation of the kynureninase gene (Kynu), whose product metabolizes neurotoxic metabolites often accumulated in PD as kynurenine. Supplementation prevented the decrease in Kynu expression induced by rotenone in the substantia nigra (p < 0.05), corroborating the behavioral data. No differences were observed concerning the methylation analysis of two CpG sites in the Kynu promoter. Instead, we suggest that folic acid and vitamin B12 increased global DNA methylation, reduced the expression of Kynu inhibitors, maintained Kynu-dependent pathway homeostasis, and prevented the memory impairment induced by rotenone. Our study raises the possibility of adjuvant therapy for PD with folic acid and vitamin B12.

Keywords: DNA methylation; Kynu; cognitive impairment; gene expression; neurodegenerative diseases; tryptophan pathway.

Figure 1

Experimental design. (A) The animals were subjected to stereotaxic surgery for rotenone or dimethylsulfoxide (DMSO) administration within the substantia nigra pars compacta. The rats were distributed into three groups: two without supplementation (WS)—rotenone (R_WS) and sham (S); one with B12 vitamin and folic acid supplementation (Sup)—rotenone (R_Sup). The animals were subjected to the object recognition task test (ORT). For molecular analysis, DNA and RNA were extracted from the substantia nigra (SN) and the striatum (St). (B) Animal numbers for each experiment. It is important to note that n = 33 corresponds to the maximum number of animals used, but only 21 samples were available in the expression analysis. Within parentheses: number of individuals for each experiment. O = object recognition task; M = methylation pattern analysis; E = gene expression analysis.

Figure 2

Exploration time of familiar and new objects in the object recognition task (ORT). The graphic shows the exploration time of each object—familiar or new—among the experimental groups. Animals from the sham group (S: F- ± 4.1, N- ± 5.2, n = 11) spent more time exploring the new object, as expected—* p < 0.05. Rotenone administration (R_WS: F- ± 6.8, N- ± 7.0, n = 10) led to an impairment in object recognition memory (p = 0.80), which was prevented in supplemented animals (R_Sup: F- ± 5.4, N- ± 8.8, n = 12)—** p < 0.01. Data are expressed as mean (SD).

Figure 3

Differences in Kynu expression between the groups in the substantia nigra and striatum. (A) Kynu expression in rotenone-treated rats without supplementation (R_WS—median: 0.006, min: 0.002, max: 0.062, n = 6) is practically nonexistent when compared to the sham group (** p < 0.05, fold change = −2.45). Those rotenone-treated rats that received the supplementation with folic acid and vitamin B12 (R_Sup—median = 1.97, min: 0.202, max: 9.05, n = 7) maintained the expression levels observed for the sham group (S—median = 1.74, min: 0.014, max: 10.49, n = 7), meaning that the rotenone-induced decrease in Kynu’s gene expression was prevented in the supplemented group (R_Sup) (* p < 0.05, fold change = −2.50). (B) There were no significant changes in Kynu expression between groups in the striatum. R_Sup—median = 4.05, min: 0.06, max: 32.06, n = 7; R_WS—median = 0.69, min: 0.03, max: 21.33, n = 7; S—median = 1.19, min: 0.02, max: 10.52, n = 7. Data are expressed as median (IQR). Min = minimum; max = maximum.

Figure 4

Effects of folic acid and vitamin B12 supplementation on Kynureninase expression. (A) In a non-pathological scenario, Kynu is possibly expressed at normal levels, as well as the repressor protein, which does not have sufficient levels to completely suppress Kynu expression. (B) In a pathological scenario, this repressor may have a hypomethylated promoter region, leading to continuous expression and increased protein levels, resulting in intense Kynu repression. KYNU can process 3-hydroxykynurenine (3-HK), and low levels of this enzyme may trigger the accumulation of 3-HK, resulting in a toxic effect due to the generation of free radical superoxide and hydrogen peroxide, which leads to cell death. (C) With folic acid and B12 supplementation, the DNA methylation process is normalized, and the repressor’s promoter region returns to its standard methylation state or becomes completely methylated, reducing or silencing the expression of this repressor and allowing for Kynu expression to occur and 3-HK to be appropriately processed.

Figure 5

The tryptophan pathway, with emphasis on the kynurenine pathway (KP) and kynureninase (KYNU) participation. The KP is responsible for catabolizing 95% of the tryptophan available in the body [42,43]. In the brain, tryptophan is catabolized into formylkynurenine by the action of indoleamine 3-dioxygenase (IDO) and later in kynurenine (KYN) by formamidase. KYN can be processed by three different enzymes, leading to other routes. If processed by kynureninase (KYNU), the formation of anthranilic acid will occur, which can later serve as a precursor for the formation of 3-hydroxyanthranilic acid (3-HAA). If KYN is catabolized by kynurenine aminotransferases (KATs), kynurenic acid (KYNA) will form. Finally, KYN can be processed by kynurenine 3-monooxygenase (KMO), resulting in 3-hydroxykynurenine (3-HK), which can be metabolized by KYNU, giving rise to 3-HAA. XA—xanthurenic acid; 3-HA—3-hydroxyanthranilic acid 3,4-dioxygenase; QUIN—acid quinolinic; TDO—tryptophan 2,3-dioxygenase.