Nasal Rifampicin Improves Cognition in a Mouse Model of Dementia with Lewy Bodies by Reducing α-Synuclein Oligomers

Abstract

α-Synuclein oligomers are thought to play an important role in the pathogenesis of dementia with Lewy bodies (DLB). There is no effective cure for DLB at present. Previously, we demonstrated that in APP- and tau-transgenic mice, oral or intranasal rifampicin reduced brain Aβ and tau oligomers and improved mouse cognition. In the present study, we expanded our research to DLB. Rifampicin was intranasally administered to 6-month-old A53T-mutant α-synuclein-transgenic mice at 0.1 mg/day for 1 month. The mice displayed memory impairment but no motor deficit at this age, indicating a suitable model of DLB. α-Synuclein pathologies were examined by the immunohistochemical/biochemical analyses of brain tissues. Cognitive function was evaluated by the Morris water maze test. Intranasal rifampicin significantly reduced the levels of [pSer129] α-synuclein in the hippocampus and α-synuclein oligomers in the visual cortex and hippocampus. The level of the presynaptic marker synaptophysin in the hippocampus was recovered to the level in non-transgenic littermates. In the Morris water maze, a significant improvement in spatial reference memory was observed in rifampicin-treated mice. Taken together with our previous findings, these results suggest that intranasal rifampicin is a promising remedy for the prevention of neurodegenerative dementia, including Alzheimer's disease, frontotemporal dementia, and DLB.

Keywords: dementia with Lewy bodies; intranasal administration; oligomer; prevention; rifampicin; α-synuclein.

Figure 1

Motor and cognitive functions in A53T-mutant α-synuclein-Tg mice. Male and female littermates were subjected to the rotarod (a), inverted screen (b), and Morris water maze test (c,d) at 6 and 9 months old (mo). The numbers of mice used are shown in each figure. (a) In the rotarod test, Tg mice first displayed a motor disturbance at 9 months. (b) This trend was confirmed by the inverted screen test using different individuals of age-matched littermates. In the Morris water maze test, memory acquisition (c) and retention (d) of Tg mice were significantly impaired as early as 6 months.

Figure 2

α-Synuclein pathologies in Tg mice. Brain sections prepared at 2, 4, 6, 8, 10, and 12 months old (mo) were stained with antibodies to [pSer129] α-synuclein (a), α-synuclein oligomers (b), and synaptophysin (c). (a) [pSer129] α-synuclein was detected in the visual cortex (VC) and hippocampal pyramidal cell layer (HC pcl) as early as 2 months and in the substantia nigra (SN) at 6 months. (b) α-Synuclein oligomers first appeared in the VC at 4 months. The accumulation became evident in the HC pcl at 6 months and in the SN at 10 months. Quantification of α-synuclein oligomers was performed in the HC and SN at 2, 6, and 10 months. Syn33 is an antibody to α-synuclein oligomers. (c) Synaptophysin in the hippocampal mossy fibers (HC mf) began to decrease at 4 months and were markedly attenuated at 6 months. Dotted lines indicate the boundaries of the HC pcl and HC mf regions. Quantification of synaptophysin was performed in the HC mf at 2, 6, and 10 months using the intensity in the HC pcl as a control.

Figure 3

Accumulation of α-synuclein oligomers in Tg mice. Brain soluble fractions were prepared at 3, 6, and 9 months old (mo) and examined for α-synuclein oligomers by Western blotting. Non-Tg littermate samples were obtained at 6 months. α-Synuclein dimers and trimers were significantly increased at 6 months. M, MagicMark XP Western Protein Standard (Invitrogen, Carlsbad, CA, USA).

Figure 4

Effect of intranasal rifampicin on the cognition of Tg mice. After the Morris water maze test in Figure 1c–d, 6-month-old (mo) Tg mice were divided into 2 groups. One group was intranasally treated with rifampicin (RFP) at 0.1 mg/day for 1 month and the other was treated with CMC. Non-Tg littermates were treated with CMC only. After treatment, a second water maze test was performed. The numbers of mice assigned are shown in each figure. (a) Rifampicin significantly improved the memory of Tg mice. The curves on the left prior to rifampicin treatment (6 months) are reproduced from the data shown in Figure 1c. (b) Memory retention was also ameliorated by rifampicin treatment.

Figure 5

Effects of intranasal rifampicin on α-synuclein pathologies in Tg mice. After the second Morris water maze test, brain sections were prepared and stained. The levels of [pSer129] α-synuclein (a) and α-synuclein oligomers (b) in the visual cortex (VC) and hippocampus (HC) were quantified. (a,b) [pSer129] α-synuclein was significantly reduced in the HC and to some extent in the VC by rifampicin treatment, while α-synuclein oligomers were significantly decreased in both regions. Syn33 is an antibody to α-synuclein oligomers. (c) The levels of activated microglia were quantified in the HC. Rifampicin significantly attenuated microglial activation. (d) Synaptophysin levels in hippocampal mossy fibers (HC mf) were measured using the intensity in the pyramidal cell layer (pcl) as a control. Rifampicin significantly recovered the synapse level in Tg mice. RFP, rifampicin; AU, arbitrary units.

Figure 6

Clearance of α-synuclein oligomers by intranasal rifampicin (RFP). After the second Morris water maze test, brain soluble fractions were prepared and subjected to Western blot analysis. The density of visualized α-synuclein bands were quantified and compared between CMC- and rifampicin-treated Tg mice. α-Synuclein monomers, dimers, and trimers were reduced by rifampicin treatment in Tg mice. + and − mean rifampicin- and CMC-treatment, respectively.