Expression Profiles of Brain-Derived Neurotrophic Factor Splice Variants in the Hippocampus of Alzheimer’s Disease Model Mouse

Abstract

Dysregulation of brain-derived neurotrophic factor (BDNF) has been implicated in Alzheimer’s disease (AD). In this study, we investigated the temporal dynamics of BDNF expression in the hippocampus of 5xFAD mice, an AD model, focusing on sex and age differences and Bdnf mRNA splice variants. At 3 months of age, female wild-type (WT) mice exhibited significantly higher Bdnf mRNA levels compared to males. However, this difference was abolished in female 5xFAD mice. At 6 months of age, no sex differences in Bdnf mRNA levels were observed in WT mice, and the levels tended to be lower in female 5xFAD mice. Additionally, a significant decrease in the mRNA levels of full-length tropomyosin-related kinase B (TrkB), a BDNF receptor, was found in female 5xFAD mice at 6 months, while mRNA levels of the truncated TrkB were increased in both male and female 5xFAD mice. Specifically, among the Bdnf mRNA splice variants, the levels of Bdnf exon IIA–IX, exon IIB–IX, exon IIC–IX, and exon IXA mRNA were significantly higher in female WT mice compared to male WT mice at 3 months, but this difference was lost in female 5xFAD mice. These findings suggest that the expression of specific Bdnf splice variants would be maintained at higher levels in the hippocampus of young female mice than in males but may be disrupted in AD model mice. Our study may provide insights into the relationship between sex differences in AD onset and BDNF expression.

INTRODUCTION

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, plays a crucial role in neuronal survival, differentiation, synaptic plasticity, and plasticity-related phenomena such as learning and memory.^1–6) For example, BDNF regulates the survival and differentiation of cortical progenitor cells,⁷⁾ increases spine density in hippocampal CA1 neurons,⁸⁾ and is essential for memory persistence.⁹⁾ BDNF expression is regulated by characteristic transcriptional and post-transcriptional mechanisms. In rodents, the Bdnf gene consists of eight untranslated exons (Bdnf exons I–VIII) and one exon containing the coding sequence of the preproBDNF protein (Bdnf exon IX).¹⁰⁾ Alternative Bdnf promoters (Bdnf promoters I–IX) are located upstream of each exon, generating multiple Bdnf mRNA splice variants that possess different types of 5′ exon but share a common 3′ exon. Additionally, there are two main polyadenylation signals, resulting in the production of Bdnf mRNA with either a long or short 3′ untranslated region (UTR). These Bdnf mRNA splice variants with different UTRs are thought to contribute to the dendritic targeting,^11,12) activity-regulated stabilization,¹³⁾ translation efficacy,^14,15) and spatiotemporal expression of Bdnf mRNA.¹⁰⁾

Due to the fundamental role of BDNF in the nervous system, alterations in its expression have been observed in patients with neurodegenerative diseases.⁶⁾ Lower levels of BDNF expression have been found in postmortem brains of patients with Alzheimer’s disease (AD).^16–18) Conversely, higher serum BDNF levels¹⁹⁾ and increased BDNF expression in the brains²⁰⁾ have been reported to be protective against aging-related cognitive decline. Shimada et al. also reported that low serum BDNF levels are associated with poorer cognitive function and mild cognitive impairment (MCI).²¹⁾ Furthermore, lower concentrations of BDNF in cerebrospinal fluid have been linked to the progression from MCI to AD.²²⁾

These findings strongly suggest that decreased BDNF expression in the brains is closely related to AD. However, it remains unclear whether this decreased expression is a cause of the disease or a consequence of neurodegeneration. Although there are fragmentary reports on changes in specific Bdnf mRNA splice variants in the brains of AD patients,²³⁾ alterations in these variants remain unclear. Additionally, AD risk is known to be higher in women.²⁴⁾ BDNF expression has been reported to be regulated by the female hormone estrogen.²⁵⁾ BDNF colocalizes with estrogen receptor α in hippocampal pyramidal neurons.²⁶⁾ Levels of Bdnf mRNA are significantly reduced by gonadectomy, and this reduction is restored by an injection of estrogen.²⁶⁾ Given several reports suggesting that estrogen may be protective against AD,^27–29) it is speculated that BDNF expression may be regulated by sex hormones, particularly in women, and that this regulatory mechanism may be abnormal in AD.

Therefore, in this study, we aimed to investigate the temporal dynamics of BDNF expression in the hippocampus of 5xFAD mice, an AD model, focusing on sex differences. 5xFAD mice carry five familial Alzheimer’s disease (FAD) mutations, which are associated with an increased risk of AD, and are widely used in AD research.³⁰⁾ Furthermore, we examined changes in the expression of Bdnf mRNA splice variants in the hippocampus of 5xFAD mice. Our findings revealed that levels of specific Bdnf splice variants in the hippocampus of female mice were higher than in males, but these higher levels of Bdnf variants were abolished in AD model mice. These results suggest sex differences in BDNF expression in the hippocampus and alterations in BDNF levels in AD, which may enhance our understanding of sex-specific risks for AD. Additionally, this study may propose the potential for therapeutic strategies targeting exon-specific BDNF expression in AD brains.

MATERIALS AND METHODS

Animals

All procedures and experiments involving animals were approved by the Animal Experiment Committee of the Takasaki University of Health and Welfare (Authorization No. 2008) and conducted in accordance with the University’s Guidelines for the Care and Use of Laboratory Animals. The mice were kept under standard laboratory conditions (12-h light/dark cycle at 22 ± 2 °C) with ad libitum access to food and water. Male 5xFAD mice (B6SJL-Tg[APPSwFlLon,PSEN1*M146L*L286V]6799Vas/Mmjax) were purchased from the Jackson Laboratory (Bar Harbor, ME, U.S.A.). 5xFAD mice carry five FAD mutations.³⁰⁾ These include three mutations in the amyloid precursor protein (APP) gene (Swedish: K670N/M671L, Florida: I716V, and London: V717I) and two mutations in the presenilin-1 (PSEN1) gene (M146L and L286V). Male 5xFAD mice were crossed with female B6SJLF1/J mice, produced by crossing C57BL/6J (B6) female and SJL/J (SJL) male mice (purchased from the Jackson Laboratory), for breeding. Littermates of wild-type (WT) mice were used as controls.

Purification of Total RNA and RT-PCR

Male and female wild-type and 5xFAD mice at 12-week-old (3 months of age) or 28-week-old (6 months of age) were deeply anesthetized with a high dose of pentobarbital (100 mg/kg body weight) and transcardially perfused with 10 mL of saline. Hippocampi were collected for total RNA extraction using ISOGEN II (Nippongene, Tokyo, Japan) according to the manufacturer’s instructions. RT-PCR was performed using PrimeScript Reverse Transcriptase (TaKaRa, Kusatsu, Japan) and SYBR Select Master Mix (ThermoFisher Scientific, Waltham, MA, U.S.A.), following the respective protocols provided by the manufacturers. Gene expression levels were quantified using the ^ΔΔCt method to calculate fold changes, with glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA serving as the endogenous control. The primer sequences used for PCR were as follows: Gapdh, 5′-GCACAGTCAAGGCCGAGAA-3′ and 5′-CTTCTCCATGGTGGTGAAGAC-3′; Bdnf, 5′-AAGGACGCGGACTTGTACAC-3′ and 5′-CGCTAATACTGTCACACACGC-3′; Ntrk2 (FL), 5′-TGTGAAGACGCTGAAGGACG-3′ and 5′-GAACTTGTTGAGGTCCCCGT-3′; Ntrk2 (T1), 5′-GGATTCTGCCTGCTGGTGAT-3′ and 5′-CAGAGTTCAGCTCACAGGG-3′; Gfap, 5′-GACAACTTTGCACAGGACCTC-3′ and 5′-ATACGCAGCCAGGTTGTTCTC-3′. The primer sequences to detect each Bdnf mRNA splice variant were as follows: exon I-S, 5′-CAAACAAGACACATTACCTTCCTGC-3′; exon IIA-S, 5′-GTGGTAGTACTTCATCCAGTTCC-3′; exon IIB-S, 5′-ACTCTTGGCAAGCTCCGGT-3′; exon IIC-S, 5′-GTAAGCCGCAAAGAAGTTCCAC-3′; exon III-S, 5′-CTCCCCGAGAGTTCCG-3′; exon IV-S, 5′-GGAAATATATAGTAAGAGTCTAGAACCTTGG-3′; exon V-S, 5′-CTCTGTGTAGTTTCATTGTGTGTTCG-3′; exon VI-S, 5′-GGACCAGAAGCGTGACAAC-3′; exon VII-S, 5′-AAAGGGTCTGCGGAACTCCA-3′; exon VIII-S, 5′-GACTGTGCATCCCAGGAGAA-3′; exon IXA-S, 5′-GGTCTGAAATTACAAGCAGATGGG-3′; exon IX-AS, 5′-GACGTTTACTTCTTTCATGGGCG-3′. A 5′ exon-specific primer and a common exon IX-AS were used to discriminate spliced Bdnf transcripts (Supplementary Fig. S1).

Measurement of BDNF Protein

Proteins were extracted using the T-PER Protein Extraction Reagent (Thermo Fisher Scientific) supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific), following the manufacturer’s instructions. Protein concentrations were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific), and the concentration was adjusted to 2 mg/mL. The amount of BDNF protein was measured using a Mature BDNF enzyme-linked immunosorbent assay (ELISA) Kit Wako, High Sensitive (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), according to the manufacturer’s instructions. The amount of BDNF protein per 1 µg of total protein was calculated.

Statistics

Data are presented as mean values ± standard error of the mean (S.E.M.). Statistical analyses described in the figure legends were performed using Prism 7 software (GraphPad, Boston, MA, U.S.A.).

RESULTS

Alterations in Bdnf mRNA Expression in the Hippocampus of Male and Female Mice

5xFAD mice express human APP and PSEN1 transgenes with five AD-related mutations. In 5xFAD mice, intraneuronal accumulation of amyloid β (Aβ) is observed from 1.5 months of age, and extracellular amyloid deposition is detected at 2 months, increased progressively with aging.³⁰⁾ Gliosis starts to be observed around 2 months and increases in parallel with amyloid deposition.³⁰⁾ Synaptic dysfunction and cognitive impairment begin to manifest from 4 to 5 months of age.^30–32) While decreased BDNF levels have been found in the hippocampus of 5xFAD mice, most studies have utilized mice aged 7–9 months or older.^33–35) Additionally, changes in the expression of multiple Bdnf mRNA splice variants in AD mouse models are poorly understood. In this study, we first examined changes in BDNF expression in the hippocampus using 3-month-old 5xFAD mice, which have been reported to exhibit early pathological and histological changes such as amyloid deposition and gliosis but no significant neuronal or cognitive impairment, as well as 6-month-old 5xFAD mice, which have been reported to be at the onset of synaptic dysfunction and cognitive decline.^30–32) It is expected to allow us to analyze the relationship between decreased BDNF expression and cognitive impairment in AD. In male WT and 5xFAD mice, no significant changes in Bdnf mRNA levels were observed at either 3 or 6 months of age (Fig. 1). In contrast, Bdnf mRNA levels in female WT mice were significantly higher than those in male WT mice at 3 months (Fig. 1A). However, Bdnf mRNA levels were significantly reduced in female 5xFAD mice compared to female WT mice, with expression levels similar to those in males (Fig. 1A). At 6 months, Bdnf mRNA expression was similar in both male and female WT mice, while female 5xFAD mice exhibited a decreasing trend in Bdnf mRNA expression (Fig. 1B). These results suggest that BDNF expression is higher in the female hippocampus than in the male hippocampus, at least at 3 months of age, but this elevation is abrogated in female 5xFAD mice. A significant genotype-by-sex interaction was observed in the mice at 3 months of age (F_{(1, 24)} = 9.291, p = 0.0055).

Fig. 1. Alterations in Bdnf mRNA Levels in the Hippocampus of Male and Female 5xFAD Mice

Changes in Bdnf mRNA levels in the hippocampus of male and female wild-type (WT) and 5xFAD mice at 3 (A) or 6 (B) months of age (12 or 28 weeks of age, respectively). The levels of all Bdnf transcripts were analyzed by PCR using primers designed for the coding region of preproBDNF protein. Changes in Bdnf mRNA levels are represented as fold-changes relative to mRNA levels in male WT mice. Means  ±  S.E.M., n = 7 (A), 6–8 (B). Data were analyzed by two-way ANOVA and the genotype-by-sex interaction was observed in the mice at 3 months of age [(A) F_{(1, 24)} = 9.291, p = 0.0055; (B) F_{(1, 24)} = 1.081, p = 0.3088]. The differences among means were analyzed using Sidak’s multiple comparisons test.

Expression of TrkB Subtypes in the Hippocampus of 5xFAD Mice

We next analyzed the changes in the expression of tropomyosin-related kinase B (TrkB), a receptor for BDNF, in the hippocampus of 5xFAD mice. TrkB has two main isoforms: full-length TrkB (TrkB-FL), which contains a tyrosine kinase domain, and truncated TrkB (TrkB-T1), which lacks this domain.³⁶⁾ We focused on the expression of these isoforms in the hippocampus of 5xFAD mice. No significant changes were observed in the expression of TrkB-FL [Ntrk2 (FL)] or TrkB-T1 [Ntrk2 (T1)] in either male or female 5xFAD mice at 3 months of age (Figs. 2A, C). Changes in Ntrk2 (FL) mRNA were slight, but seemed to have a similar trend to the changes in Bdnf mRNA levels (Figs. 1A, 2A). A significant genotype-by-sex interaction was observed in Ntrk2 (FL) mRNA at 3 months of age (F_{(1, 24)} = 5.621, p = 0.0261). Ntrk2 (FL) mRNA expression was significantly decreased in the hippocampus of female 5xFAD mice at 6 months (Fig. 2B). In contrast, Ntrk2 (T1) mRNA expression increased in both male and female 5xFAD mice at 6 months (Fig. 2D).

Fig. 2. Alterations in mRNA Levels of TrkB Subtypes in the Hippocampus of Male and Female 5xFAD Mice

Changes in the mRNA levels of TrkB-FL [A, B; Ntrk2 (FL)] and TrkB-T1 [C, D; Ntrk2 (T1)] in the hippocampus of male and female WT and 5xFAD mice at 3 (A, C) and 6 (B, D) months of age. Changes in mRNA levels are represented as fold-changes relative to mRNA levels in male WT mice. Means  ±  S.E.M., n = 7 (A, C), 6–8 (B, D). Data were analyzed by two-way ANOVA and the genotype-by-sex interaction was observed in Ntrk2 (FL) mRNA in the mice at 3 months of age [(A) F_{(1, 24)} = 5.621, p = 0.0261; (B) F_{(1, 24)} = 4.1, p = 0.0542; (C) F_{(1, 24)} = 0.4529, p = 0.5074; (D) F_{(1, 24)} = 0.1044, p = 0.7494]. The differences among means were analyzed using Sidak’s multiple comparisons test.

TrkB-FL and TrkB-T1 are predominantly expressed in neurons and astrocytes, respectively.³⁷⁾ Given that gliosis begins around 2 months in 5xFAD mice,³⁰⁾ we hypothesized that the increase in Ntrk2 (T1) mRNA expression was due to the rise in glial cells such as astrocytes. The expression of glial fibrillary acidic protein (Gfap), an astrocyte marker, was significantly elevated in the hippocampus of both male and female 5xFAD mice at 3 months, with a more pronounced increase at 6 months (Figs. 3A, B). These findings suggest that the increase in TrkB-T1 expression is linked to the proliferation of astrocytes in 5xFAD mice. Supporting this, Ntrk2 (T1) mRNA levels showed strong correlations with Gfap mRNA levels in the hippocampus of 6-month-old male and female 5xFAD mice [Figs. 3C, D; correlation coefficient (r) = 0.4129 (male WT), 0.8915 (male 5xFAD), 0.3755 (female WT), and 0.6171 (female 5xFAD), respectively].

Fig. 3. Alterations in Gfap mRNA Levels in the Hippocampus of Male and Female 5xFAD Mice

(A, B) Changes in Gfap mRNA levels in the hippocampus of male and female WT and 5xFAD mice at 3 (A) or 6 (B) months of age. Changes in mRNA levels are represented as fold-changes relative to mRNA levels in male WT mice. Means  ±  S.E.M., n = 7 (A), 6–8 (B). Data were analyzed by two-way ANOVA and the genotype-by-sex interaction was not observed [(A) F_{(1, 24)} = 0.2597, p = 0.6150; (B) F_{(1, 24)} = 0.0006375, p = 0.9801]. The differences among means were analyzed using Sidak’s multiple comparisons test. (C, D) Correlation between mRNA levels of Gfap and Ntrk2 (T1) in the hippocampus of male (C) and female (D) WT and 5xFAD mice at 6 months of age. Statistical analysis of correlation (Pearson’s correlation) was conducted.

Expression of Bdnf mRNA Splice Variants in the Hippocampus of 5xFAD Mice

We next focused on the Bdnf mRNA splice variants (Fig. 4A) altered in the hippocampus of 5xFAD mice at 3 months. First, we compared the expression levels of each Bdnf splice variant in male and female WT mice (Fig. 4B). Bdnf exon V–IX and exon VII–IX were expressed at very low levels in the hippocampus and were difficult to detect (data not shown). Relative to Bdnf exon I–IX, Bdnf exon III–IX and exon VIII–IX exhibited low but detectable expression levels (Fig. 4B). In the mouse hippocampus, Bdnf exon I–IX, exon IIC–IX, exon VI–IX, and exon IXA were predominantly expressed, with Bdnf exon IV–IX also showing moderate expression (Fig. 4B). Compared to these variants, the expression levels of Bdnf exon IIA–IX and exon IIB–IX were lower but still detectable (Fig. 4B).

Fig. 4. Comparison of Relative Expression Levels of Bdnf mRNA Splice Variants in the Hippocampus of Male and Female WT Mice at 3 Months of Age

(A) Schematic of the mouse Bdnf gene structure and Bdnf mRNA splice variants. (B) Relative expression levels of Bdnf mRNA splice variants in the hippocampus of male and female WT mice at 3 months of age. Changes in mRNA levels are represented as a percentage relative to the levels of Bdnf exon I–IX mRNA in male WT mice. Means  ±  S.E.M., n = 7.

We then investigated changes in the levels of Bdnf splice variants in WT and 5xFAD mice. In male WT and 5xFAD mice, no significant alterations in Bdnf splice variants were observed, though there was a trend toward lower levels of Bdnf exon I–IX, exon IIB–IX, and exon III–IX in male 5xFAD mice (Fig. 5). In contrast, Bdnf exon IIA–IX, exon IIB–IX, exon IIC–IX, and exon IXA were significantly more highly expressed in female WT mice compared to male WT mice (Fig. 5). However, the elevated levels of these variants in female WT mice were abolished in female 5xFAD mice (Fig. 5). Specifically, Bdnf exon I–IX, exon IIA–IX, exon IIB–IX, and exon IIC–IX were significantly reduced in the hippocampus of female 5xFAD mice (Fig. 5). A significant genotype-by-sex interaction was observed in Bdnf exon IIA–IX at 3 months of age (F_{(1, 24)} = 4.499, p = 0.0444). Statistical analysis also showed the significant effects of sex on Bdnf exon IIA–IX, exon IIB–IX, exon IIC–IX, and exon IXA [Bdnf exon IIA–IX mRNA (F_{(1, 24)} = 6.156, p = 0.0205), Bdnf exon IIB–IX mRNA (F_{(1, 24)} = 4.74, p = 0.0395), Bdnf exon IIC–IX mRNA (F_{(1, 24)} = 10.14, p = 0.0040), and Bdnf exon IXA mRNA (F_{(1, 24)} = 7.594, p = 0.0110)].

Fig. 5. Alterations in Levels of Bdnf mRNA Splice Variants in the Hippocampus of Male and Female 5xFAD Mice at 3 Months of Age

Changes in levels of Bdnf mRNA splice variants in the hippocampus of male and female WT and 5xFAD mice at 3 months of age. Changes in mRNA levels are represented as fold-changes relative to mRNA levels in male WT mice. Means  ±  S.E.M., n = 7. Data were analyzed by two-way ANOVA and the genotype-by-sex interaction was observed in Bdnf exon IIA–IX mRNA [(exon I–IX) F_{(1, 24)} = 1.667, p = 0.2089; (exon IIA–IX) F_{(1, 24)} = 4.499, p = 0.0444; (exon IIB–IX) F_{(1, 24)} = 3.191, p = 0.0867; (exon IIC–IX) F_{(1, 24)} = 3.582, p = 0.0705; (exon III–IX) F_{(1, 24)} = 0.153, p = 0. 6992; (exon IV–IX) F_{(1, 24)} = 1.069, p = 0.3115; (exon VI–IX) F_{(1, 24)} = 0.3021, p = 0.5876; (exon VIII–IX) F_{(1, 24)} = 0.01955, p = 0.8900; (exon IXA) F_{(1, 24)} = 3.69, p = 0.0667]. The differences among means were analyzed using Tukey’s multiple comparisons test was performed.

Lastly, we examined BDNF protein expression in the hippocampus of 5xFAD mice at 3 months. Compared to male WT mice, BDNF protein levels tended to be higher in female WT mice, but this was reduced in female 5xFAD mice (Fig. 6A). These changes in BDNF protein levels likely correspond to the changes in Bdnf mRNA levels (Fig. 1A). Supporting this, BDNF protein levels were significantly correlated with Bdnf mRNA levels (Fig. 6B). Taken together, our findings suggest that specific Bdnf mRNA splice variants are more highly expressed in the female hippocampus compared to males, and this regulatory mechanism is impaired in 5xFAD mice.

Fig. 6. Alterations in BDNF Protein Levels in the Hippocampus of Male and Female 5xFAD Mice at 3 Months of Age

(A) Changes in BDNF protein levels in the hippocampus of male and female WT and 5xFAD mice at 3 months of age. The amount of BDNF protein is represented as the amounts per 1 µg total protein. Means  ±  S.E.M., n = 7. Data were analyzed by two-way ANOVA and the genotype-by-sex interaction was not observed (F_{(1, 24)} = 1.642, p = 0.2123), while the effect of genotype was significantly observed (F_{(1, 24)} = 4.493, p = 0.0466). (B) Correlation between Bdnf mRNA levels and BDNF protein levels in the hippocampus of male and female WT and 5xFAD mice at 3 months of age. Statistical analysis of correlation (Pearson’s correlation) was conducted.

DISCUSSION

Although BDNF expression is reduced in the brains of AD patients, it remains unclear whether this decrease is a cause or consequence of disease progression. Additionally, sex differences in AD risk have been observed, however, the association between sex differences in BDNF expression and AD is not fully understood. This study demonstrated that the expression of specific Bdnf mRNA splice variants in the female hippocampus was higher than in males, and these elevated variants were reduced in female 5xFAD mice.

Bdnf mRNA levels were significantly higher in the hippocampus of female WT mice compared to males, possibly due to the effects of estrogen on BDNF expression.²⁵⁾ Indeed, estrogen upregulates Bdnf mRNA levels in cultured neurons and in the hippocampus of ovariectomized animals.^25,38,39) Higher BDNF levels in females than males have been reported in several regions, including the hippocampus, of rat brain.⁴⁰⁾ Sex differences in BDNF expression in the mouse brain remain controversial,⁴⁰⁾ though one study suggests that BDNF protein levels in the hippocampus are higher in female mice.⁴¹⁾ Moreover, we showed that specific Bdnf splice variants, such as Bdnf exon IIA–IX, exon IIB–IX, exon IIC–IX, and exon IXA, were significantly more highly expressed in female WT mice (Fig. 7A). Functional estrogen response elements (EREs) have been identified in Bdnf promoter IX located upstream of Bdnf exon IXA.³⁹⁾ Analyses using ChIP Atlas⁴²⁾ suggest that estrogen receptor α may bind downstream of Bdnf exon III in analyses using estrogen receptor-expressing fibroblasts (Supplementary Fig. S2). These suggest that estrogen activate specific Bdnf promoters, possibly explaining the higher expression of specific Bdnf mRNA splice variants in females (Fig. 7B). A previous study has shown significant reductions in mRNA and protein levels of aromatase, an estrogen synthetase, in the hippocampus of female 5xFAD mice at 3 months of age.⁴³⁾ Reduced aromatase and brain estrogen levels have also been reported in AD patients.⁴⁴⁾ Thus, estrogen likely regulates specific Bdnf promoters in the female brain, and this regulation may be disrupted in AD brains (Fig. 7B). Since significant reductions in BDNF levels have been observed in the hippocampus of other female AD mouse models, such as APP/PS1 and App^NL-G-F knock-in mice,^45,46) it will be important to investigate whether sex differences observed in this study are also present in these AD models.

Fig. 7. Summary of Alterations in 5′ Exon-Specific Bdnf mRNA and Possible Schematic Model of Splice Variant-Selective Alterations in Bdnf mRNA in the Hippocampus of 5xFAD Mice

(A) Summary of changes in the expression of 5′ exon-specific Bdnf mRNA in the hippocampus of male and female 5xFAD mice at 3 months of age. Based on the results in Figs. 4B and 5, the relative expression levels among each variant and changes in the levels of Bdnf mRNA splice variants in male and female WT and 5xFAD mice are shown in pseudocolor. Values in each box show percentages relative to the levels of Bdnf exon I–IX mRNA in male WT mice and fold-changes relative to the levels of each variant in male WT mice, respectively. In addition, based on a previous report,⁵⁹⁾ differences in the translation efficiency of each variant are also included. ^(#) Another report suggested the presence of an initiation codon with high translation efficiency on the 3′ side of Bdnf exon I,¹⁴⁾ contributing to efficient BDNF translation. (B) A schematic representation of the regulation of Bdnf expression in the hippocampus of WT and 5xFAD mice. The expression of specific variants, particularly exon II- and exon IXA-containing variants, may be regulated by estrogen-dependent transcription of Bdnf gene, and this regulatory mechanism may be disrupted in AD brains. In addition, the levels of Bdnf exon I–IX, exon II–IX, and exon III–IX tended to be downregulated in males as well as females, suggesting that transcriptional regulatory mechanisms that modulate the expression of Bdnf exon I–IX, exon II–IX, and exon III–IX, such as Bdnf promoter I and intronic enhancer, may be disrupted by Aβ.

No significant changes in BDNF levels were observed in male mice. However, an analysis of Bdnf splice variants showed a trend toward reduced levels of variants including Bdnf exon I–IX, exon IIB–IX, and exon III–IX in the hippocampus of male 5xFAD mice, which was also observed in females (Fig. 7A). The reduction of Bdnf exon I and exon II has been also reported in the postmortem brains of AD patients, while the expression of Bdnf exon IV (formerly exon III) was also reduced in these advanced AD brains.⁴⁷⁾ Based on our findings, the expression of Bdnf exon I–IX, exon II–IX, and exon III–IX may be preferentially reduced in brains with early AD, whereas the expression of other variants like Bdnf exon IV–IX may decline in brains with advanced and severe AD. Because Bdnf exons I, II, and III are located close to each other in genomic DNA, it has been suggested that the expression of the BDNF gene containing these exons may be regulated via a common transcriptional regulatory mechanism.⁴⁸⁾ Supporting this, Maynard et al. have shown that selective disruption of BDNF expression from Bdnf promoter I not only abolished the expression of Bdnf exon I–IX mRNA but also reduced the expression of Bdnf exon II–IX mRNA.⁴⁹⁾ Additionally, Tuvikene et al. reported that a novel intronic enhancer located downstream of exon III activates the expression of Bdnf exon I–IX, exon II–IX, and exon III–IX.⁴⁸⁾ Considering these reports, it is suggested that the transcriptional machinery commonly involved in the regulation of Bdnf exon I–IX, exon II–IX, and exon III–IX expression is disrupted relatively earlier in the hippocampus of 5xFAD mice (Fig. 7B), resulting in the splice variant-selective reduction of Bdnf mRNA. Aβ42 has been reported to disrupt the transcription of cAMP response element-binding protein (CREB) target genes including Bdnf.^50–52) Because CREB-binding sites are present in the Bdnf promoter I and intronic enhancer,^48,53) higher levels of Aβ42 in the 5xFAD brains would disrupt CREB-dependent transcription, resulting in decreased BDNF expression. The precise molecular mechanism underlying the selective alterations in the splice variants of Bdnf mRNA expression remains to be elucidated.

We also found that the levels of TrkB-FL and BDNF decreased in the hippocampus of female 5xFAD mice at 6 months, while TrkB-T1 levels significantly increased in both sexes. The increase in TrkB-T1 could be due to astrocyte proliferation, as increases in Ntrk2 (T1) and Gfap mRNA were correlated in the hippocampus of 5xFAD mice. However, although Gfap mRNA levels increased significantly in the hippocampus of 5xFAD mice at 3 months of age, Ntrk2 (T1) levels remained unchanged. In 6-month-old 5xFAD mice, Gfap mRNA levels was elevated 5- to 6-fold, while Ntrk2 (T1) mRNA levels increased only 1.5-fold. Therefore, the increase in astrocytes may not be sufficient to explain the increase in TrkB-T1 in 3-month-old 5xFAD mice. However, a previous in vitro study indicates that Aβ peptide exposure increased in TrkB-T1 levels even when glial cells including astrocytes were reduced,⁵⁴⁾ suggesting that the increases in TrkB-T1 levels might occur independently of gliosis in 5xFAD mice. Similar alterations in the expression of BDNF and TrkB have been reported in the brains of AD patients,^16,17,55,56) strongly suggesting impairment of BDNF/TrkB signaling in the AD brains. Since TrkB-T1 was originally identified as an endogenous dominant-negative form of TrkB-FL,⁵⁷⁾ the observed decreased TrkB-FL and increased TrkB-T1 in the hippocampus of 6-month-old female 5xFAD mice could disrupt normal BDNF/TrkB signaling, resulting in the acceleration of cognitive impairment and possibly neurodegeneration. In the present study, although TrkB-FL levels remained unchanged in male 5xFAD mice, the increase in TrkB-T1 levels suggests that the balance between TrkB-FL and TrkB-T1 may also be disturbed in the male hippocampus.

In this study, we demonstrated exon-selective alterations in Bdnf mRNA splice variants in the hippocampus of 3-month-old female 5xFAD mice, which have been reported to show pathological and histological abnormalities without noticeable neuronal and cognitive impairment.^30–32) This sex-specific reduction in BDNF expression in the hippocampus of 5xFAD mice may contribute to the earlier impairment of spatial memory observed in female 5xFAD mice.⁵⁸⁾ The Bdnf exon I–IX mRNA was highly enriched in synaptoneurosomes compared to other variants.¹⁵⁾ Vaghi et al. reported that 5′ UTR of each Bdnf splice variant exhibits a distinct translation ability⁵⁹⁾ (Fig. 7A). Furthermore, the 3′ end of Bdnf exon I contains an initiation codon that is not present in other exons, and it is suggested that this contributes to efficient BDNF translation.¹⁴⁾ Thus, the alterations in specific Bdnf splice variants observed in this study may affect BDNF protein levels. Additionally, the 5′ UTR of Bdnf transcripts is involved in dendritic targeting of Bdnf mRNA; specifically, Bdnf exon II–IX significantly facilitates dendritic targeting of the mRNA.¹²⁾ Furthermore, BDNF derived from Bdnf promoter I has been implicated in activity-regulated structural plasticity.⁶⁰⁾ Recently, Bach et al. showed that the upregulation of Bdnf exon I–IX mRNA specifically regulates genes associated with dendritic growth and enhances the density of mushroom spines.⁶¹⁾ These reports strongly suggest that Bdnf exon I–IX and exon II–IX would particularly contribute to the fine-tuning of synaptic plasticity, and their dysregulation in the hippocampus of female 5xFAD mice may result in synaptic dysfunction and memory impairment in AD. Our study indicates that sex differences in BDNF levels may be involved in sex-specific AD risk, potentially linked to changes in estrogen levels in AD. Additionally, our study also proposes the possibility of developing therapeutic strategies targeting exon-specific BDNF expression.

Acknowledgments

This work was supported by the Japan Society for the Promotion of Science (JSPS), under KAKENHI Grant Numbers: 16H05275 (Grant-in-Aid for Scientific Research [B] to M.F.) and 22K11859 (Grant-in-Aid for Scientific Research (C) to M.F.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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