Minocycline prevents early age-related cognitive decline in a mouse model of intellectual disability caused by ZBTB18/RP58 haploinsufficiency

doi:10.21203/rs.3.rs-3992401/v1

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Minocycline prevents early age-related cognitive decline in a mouse model of intellectual disability caused by ZBTB18/RP58 haploinsufficiency

https://doi.org/10.21203/rs.3.rs-3992401/v1

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published 12 Oct, 2024

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Transcriptional repressor ZBTB18/RP58 haploinsufficiency, which comprises heterozygous ZBTB18/RP58 missense and truncating variants, causes intellectual disability. However, the mechanism responsible for the onset of intellectual disability, and whether this disorder can be prevented and treated, are unknown. We focused on changes in cognitive function about adulthood in intellectual disability. Heterozygous Zbtb18/Rp58-knockout mice were used as a model for ZBTB18/RP58 haploinsufficiency. We assessed multiple behaviors and local field potential, examined DNA fragmentation and mitochondrial morphology, and performed histochemical analysis and transcriptome analysis in the hippocampus to evaluate chronic inflammation. Object location memory assessed by an object location test in wild-type mice was the same in 2 and 4–5-month-old mice, but it was impaired in 12–18-month-old mice. By contrast, object location memory in 2-month-old Zbtb18/Rp58 heterozygous-knockout mice was similar to that in age-matched wild-type mice but was impaired by 4–5-months of age, indicating the early onset of impaired spatial memory in the mutant mice. Zbtb18/Rp58 heterozygous-knockout mice exhibited early onset of DNA damage accumulation and an increment of activated microglia in the dentate gyrus that was associated with defective DNA repair. Because dementia is an established comorbidity in persons with intellectual disability, mutant mice may have mimicked not only delay of cognitive function but also an acquired cognitive impairment. Notably, chronic minocycline therapy, which has neuroprotective and anti-inflammatory effects, attenuated age-related phenotypes, such as accumulation of DNA damage, an increase of microglial activation and impairment of object location memory, in Zbtb18/Rp58 heterozygous-knockout mice. In conclusion, Zbtb18/Rp58 heterozygous-knockout mice showed early onset of cognitive decline caused by inflammation with a defect in DNA repair. Minocycline prevented early-onset cognitive decline, suggesting that it could have potential as a therapeutic agent for the treatment of ZBTB18/RP58 haploinsufficiency-associated cognitive dysfunction.

intellectual disability

cognitive decline

DNA damage

DNA repair

inflammation

ZBTB18/RP58

Intellectual disability (ID) is a neurodevelopmental disorder in which deficits in intellectual and adaptive function begin during development (1). Approximately 1% of the general population have ID. The causes of ID include genetic, environmental, trauma, neurological, nutritional, and metabolic disorders. Genetic factors are major contributors to ID (2). The 1q43q44 deletion syndrome also causes ID, and Zinc finger and BTB domain-containing protein 18(ZBTB18)/RP58 (hereafter referred to as RP58) has been identified as one of the gene responsible for 1q43q44 deletion syndrome (3). RP58, also known as ZBTB18, ZNF238, and ZFP238, is a transcriptional repressor protein with a BTB domain in its N-terminus and four Zn finger motifs in its C-terminus (4). In fact, truncating variants of RP58 are found in individuals with ID (4), indicating that haploinsufficiency of RP58 causes ID. Considering that genome sequencing of individuals with ID has revealed many nonsense and missense mutations in the RP58 gene (5, 6), abnormality of RP58 may lead to various neurodevelopmental disorders (7), and it is important to establish a strategy for therapy of RP58 haploinsufficiency.

We have previously generated Rp58 conventional and conditional knockout (KO) mice and showed that RP58 plays a critical role in the formation of the cerebral cortex (8–13). RP58 is highly expressed in the neonatal brain and its expression continues after brain maturation (14). Rp58 homozygous-KO mice exhibit a lethal phenotype immediately after birth, whereas Rp58 heterozygous knockout mice (Rp58 hetero-KO mice) are macroscopically indistinguishable from wild-type mice after birth (8). RP58 expression continues into adulthood in the glutamatergic neurons of the cerebral cortex (14), but its postnatal role is unknown. In our previous paper, we showed that RP58 heterozygous-knockout mice can serve as a model for RP58 haploinsufficiency (15). However, it is unclear why aberrations in RP58 cause ID. Moreover, there is no known prevention or treatment strategy for this disorder.

It is well known that ID is associated with a susceptibility to dementia (56, 57). In this study, we focused on cognitive dysfunction in ID in adulthood using Rp58 hetero-KO mice as a model of ID associated with RP58 haploinsufficiency. Interestingly, Rp58 hetero-KO mice showed impaired spatial cognition in adulthood, suggesting that these mice could serve as a model for early cognitive decline in ID. At the same time, we observed that Rp58 hetero-KO mice exhibit early accumulation of DNA damage caused by defective DNA repair. Inflammation was also observed along with the accumulation of DNA damage in Rp58 hetero-KO mice. These results suggest that the age-related cognitive impairment associated with RP58 haploinsufficiency caused by neuroinflammation associated with defective DNA repair. The central nervous system is particularly susceptible to DNA damage repair defects, leading to various neurodevelopmental and neurodegenerative disorders, including ID (16). The damaged DNA drives activation of the cGAS-STING signaling pathway, which induce immune response (17). These facts may explain the role of RP58 in brain. Therefore, DNA damage- and inflammation-targeted treatment is presumed to be effective in pathologies associated with RP58 haploinsufficiency. Interestingly, we found that minocycline, which has neuroprotective and anti-inflammatory effects, (18, 19) attenuated the age-related phenotype of Rp58 hetero-KO mice, suggesting that it could form the basis of a strategy for the therapy of RP58 haploinsufficiency-associated cognitive dysfunction.

All experimental procedures were approved by the Animal Experimentation Ethics Committee of the Tokyo Metropolitan Institute of Medical Science (49040).

Experimental model

Animals

The original lines of RP58 mutant mice (Rp58 homo-KO mice) were established as a C57/BL6 congenic line (a genetic background strain) through repeated back-crosses, as described previously (8). Since Rp58 homo-KO mice die immediately after birth, Rp58 hetero-KO mice and wild-type mice were used to investigate the pathogenesis of RP58 haploinsufficiency. All mice were maintained under a 12:12 h light/dark cycle (lights on at 8:00 AM). Efforts were made to minimize the number and suffering of animals used, in accordance with the 3Rs principle of reduction, replacement, and refinement, as stipulated by the Animal Welfare and Control Law. Experiments were performed during adolescence (young) stages (2 months), adulthood (4–5 months), and old age (12–18 months) (Fig. 1A). The influence of the menstrual cycle could not be ruled out in female mice; therefore, behavioral assessments were performed using male mice. For example, female mice in estrus or proestrus explore relocated objects more than female mice in diestrus (20–22).

Behavioral assessments

Male mice were habituated to a behavioral room for at least 1 h prior to the start of the behavioral test. Mice that displayed outlier behavior, that is, greater or less than two standard deviations from the mean, were not included in behavioral analysis (3–10 in each group).

The object location test consisted of three phases as described previously (23). All phases were performed under a light intensity of 10 lux. On day 1, the animals were placed into an empty square box (50 × 50 cm²) for 10 min. On day 2, the animals were placed into the same box for 10 min with two identical, unfamiliar, objects. On day 3, the animals were placed into the same box, in which one of the two objects were displaced to a novel location. The discrimination index was calculated as the ratio of the time the animal spent exploring the novel place to the time they spent exploring the familiar place for 10 min: discrimination index = (time exploring the novel place - time exploring the familiar place)/(time exploring the novel place + time exploring the familiar place). Every day, the exploration time in each place was calculated automatically using a DVTrack video tracking system (Muromachi, Tokyo, Japan).

For the open field test, each mouse was placed in the center of the apparatus (50 × 50 cm²) and was allowed to move freely for 30 min. All phases were performed under a light intensity of 10 lux. Horizontal activity was collected every 10 min using the DVTrack video tracking system (Muromachi, Tokyo, Japan).

The rotarod test consisted of five trials with 1 h intervals between trials. Prior to the test, to habituate the mice to the rotarod, the animals were placed for five minutes on a drum (3 cm diameter; MK-660B; Muromachi Kikai Co., Ltd., Tokyo, Japan) that was rotating at 1- and 4 rpm. An accelerated rotarod test was used to assess motor learning ability from 1–40 rpm over a period of five minutes.

The fear conditioning test consisted of three phases as described previously (23). On day one, the animals were placed into a triangular box for 5 min under a light intensity of 30 lux. On day 2, the animals were placed into a square box with a stainless-steel grid floor and were allowed to explore the box freely for 2 minutes. Subsequently, a tone, which served as the conditioned stimulus (CS), was presented for 30 sec. During the last 1 sec of the presentation of the CS, a 0.75 mA electric shock was applied, which served as the un-conditioning stimulus (US). Two more CS-US parings were presented with a 1-minute inter-stimulus interval under a light intensity of 100 lux. On day 3, the animals were placed into the same square box as used on day 2 for 5 minutes under a light intensity of 100 lux. In each test, the percentage of freezing time and distance traveled (cm) were calculated automatically using ImageFZ software (O’Hara & Co., Tokyo, Japan). Freezing was taken to indicate that the mice remembered the box (context) in which they were exposed to the electrical shock.

Electrophysiology

Mice were anesthetized with isoflurane (1.5%) and implanted with a custom-designed electrode comprising two tetrodes in the CA1 area of the hippocampus (1.8 mm anterior to bregma, 1.4 mm lateral to the midline, and 1.2 mm from the brain surface). Individual tetrodes consisted of four twisted polyimide-coated tungsten wires (single wire diameter, 12.7 µm; California Fine Wire, Grover Beach, CA). One additional screw was threaded into the bone above the cerebellum as a reference and for grounding. The electrodes were connected to an electrode interface board (EIB-18, Neuralynx, MT) on a microdrive. Behavioral assessments were performed at least one week after surgery.

Local field potential (LFP) recordings at a sampling rate of 30 kHz were obtained with open-source hardware (Open Ephys, Cambridge, MA) while mice were engaged in the object location test. For synchronization with behavioral data, a transistor-transistor logic pulse was used. After the recordings, electrode lesions were induced with direct current (20 µA) stimulation for 10 sec using one of the four tetrode leads.

Analysis of electrophysiology data

All data analyses were performed using the built-in software in MATLAB (MathWorks, Inc., MA). LFPs were down-sampled to 1,000 Hz and the data were extracted 1 sec before and after contact with the objects. Theta power was calculated by recording the mean value of the power spectrum between 4 and 8 Hz. This value was normalized to the theta power of mice during walking.

Immunohistochemistry

Male and female mice in adolescence, adulthood, or old age were anesthetized with isoflurane, and sequentially transcardially perfused with phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA) in PBS. The brains were removed, post-fixed, cryoprotected in 20% sucrose overnight and then in 30% sucrose for two days at 4℃, embedded in optimal cutting temperature compound, and frozen at -30℃. Coronal sections (20 µm-thick) of mouse brains were then obtained with a cryostat. Free-floating sections containing the hippocampus were incubated in HistoVT One (Nakalai, Kyoto, Japan) at 70 ℃ for 20 min, incubated in 0.4% Block Ace diluted in PBS containing 0.2% TritonX (DS Pharma Biomedical, Osaka, Japan) in PBS at room temperature (RT) for 20 min, and then incubated with primary antibodies (Supplemental Table 1). Primary antibodies were diluted 1:500 in PBS containing 0.3% Triton X-100. The sections were then incubated with secondary antibodies (Jackson, ME) (diluted 1:500 in PBS containing 0.3% Triton X-100).

For GluR2, Rp58, and parvalbumin (PV) triple staining, tissue sections (50 µm thick) were incubated at RT for 20 minutes in 1% H₂O₂ after incubation with HistoVT. After further incubation in PBS containing 1% BlocAce and 0.2% TritonX100, sections were incubated with rabbit anti-GluR2 (1:500), chicken anti-RP58 (1:1000), and goat anti-PV antibody (diluted 1:1000 in PBS containing 0.4% BlocAce) and then incubated with secondary antibody (diluted 1:1000 in PBS). To enhance the GluR2 signal, a biotinylated secondary antibody was reacted with the GluR2 antibody, and the biotin was labeled with horseradish peroxidase (HRP) using the VACTASTAIN Elite ABC-HRP Kit (Vector #PK-6100). GluR2 was detected using Tyramide Signal Amplification (TSA) Cyanine 3 (Perkin Elmer #FP1046). The tissue sections were incubated with DAPI (Nacalai Tesque, Kyoto, Japan) for nuclear staining. Micrographs of immunofluorescence-stained sections were captured and digitized using a FluoView® FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan).

Analysis of immunostaining

To quantify single-stranded DNA (ssDNA) and gamma H2AX in cells, the number of cells with five or more nuclear ssDNA foci or gamma H2AX foci was counted (24). For the analysis of microglia states, immunostaining with an antibody against CD68 was performed. CD68 is a lysosomal-associated protein found in macrophages/microglia and is associated with phagocytic cells. An Iba1 antibody was used as a marker for both resting and activated microglia. Microglia with aggregated CD68 expression were categorized as reactive, and the percentage of reactive microglia was compared between groups (25, 26). Antibody information is provided in the Key Resources table.

Irradiation and immunohistochemistry

Animals were irradiated with a dose of 0.3 Gray. At 1-, 6-, or 24 h after irradiation, mice were anesthetized with isoflurane and transcardially perfused with PBS followed by 4% PFA in PBS. Subsequent steps were similar to those described earlier under immunohistochemistry.

Electron microscopy

Adult male and female mice were anesthetized with isoflurane and transcardially perfused with saline followed by 2% PFA and 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4). The brains were removed, post-fixed, and coronal sections of the mouse brain containing the dentate gyrus were then obtained with a VT1200S microtome (300 µm) (Leica, Wetzlar, Germany). Micro-dissected areas were post-fixed for 2 h at 4 ℃ in 2% osmium tetroxide in 0.1 mol/L cacodylate buffer. Tissue blocks were dehydrated sequentially in 50% ethanol for 5 min, 70% ethanol for 15 min, 80% ethanol for 15 min, 90% ethanol for 15 min, and in 100% ethanol at RT for 20 min. The tissues were then subsequently infiltrated with propylene oxide three times for 10 min each, treated with a 1:1 solution of propylene oxide:epoxy resin for 2 h, and then incubated in epoxy resin overnight. Each block was placed flat on glass microscope slides and horizontally mounted on gelatin capsules (Lilly, IN). After embedding, each block was polymerized in epoxy resin (EPON 812, TAAB, Berkshire, England) at 60°C for 48 h. Polymerized blocks were trimmed to a section containing the dentate gyrus using an ultramicrotome PowerTomeX (RMC Boeckeler, AZ). Semi-thin (1 µm) sections were stained with toluidine blue and used to guide further cutting of the specimen block to ultra-thin sections (50–80 nm). Ultra-thin sections were placed in formvar-coated single-slot grids. After staining with uranyl acetate and lead citrate, ultra-thin sections were observed under a JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan) equipped with a bottom-mounted charge-coupled device camera and subsequently processed using ImageJ.

Quantification of ultrastructural defects: Quantification of mitochondrial ultrastructural defects was performed on transmission electron microscopy images acquired at a magnification of 10,000×. Although mitochondria are sparse within astrocytic processes of the dentate gyrus, the criteria used by Sisková et al. (27) for the identification of astrocytes and neuronal cells were applied to ensure that predominantly, if not exclusively, neuronal mitochondria were observed. Every mitochondrion of neuronal origin was analyzed and, based on its ultrastructural appearance, classified into one of the following categories according to Sisková et al. (28): intact mitochondria with normal-appearing cristae (Type I); abnormal mitochondria with either swollen, irregular, or whirling cristae (Type II); mitochondria with a discontinuous outer membrane or deficient cristae (Type III); and mitochondria with both swollen and deficient cristae or with both a discontinuous outer membrane and swollen cristae (Type IV). At least 14 mitochondria from each region were analyzed per animal.

Drug treatment

Minocycline was administered in drinking water to wild-type and Rp58 hetero-KO mice from weaning to the day before the behavioral test or perfusion. Minocycline was dissolved at a concentration of 0.015 mg/mL in filtered water.

Transcriptome analysis

Three independent total RNA samples from hippocampi of mice in each group were mixed and purified using a RNeasy Plus Universal Kit (Qiagen, Hilden, Germany). RNA quality was assessed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Cy3-labeled complementary RNA was prepared using a Low Input Quick Amp Labeling Kit (Agilent Technologies) in accordance with the manufacturer’s protocol. Samples were hybridized to the SurePrint G3 Mouse Gene Expression v2 Microarray (G4852B; Agilent Technologies). Thereafter, the array was washed and scanned using the SureScan Microarray Scanner (Agilent Technologies). Microarray images were analyzed using Agilent’s Feature Extraction software (Agilent Technologies) with default settings for all parameters. Data from each microarray analysis were normalized by shifting to the 75th percentile without baseline transformation. Microarray results were deposited in the Gene Expression Omnibus database under the accession number GSE207955.

Statistical analysis

Group data are presented as the mean ± SEM. The statistical significance of between-group differences was assessed by the one sample t-test for the object location test, a two-way repeated ANOVA followed by Holm’s test for the open field, rotarod, and fear conditioning tests, Dunnett’s test for immunostaining, Pearson’s chi-squared test for electron microscopy, and the paired t-test for theta power in the hippocampal CA1 region, using JMP software (SUS Institute, NC).

Rp58 heterozygous-KO mice exhibit early-onset cognitive decline

We first investigated whether Rp58 hetero-KO mice, which we used as a model of RP58 haploinsufficiency, had behavioral deficits by conducting multiple behavioral tests in adulthood. Four to five-month-old Rp58 hetero-KO mice did not show significant behavioral changes in the open field, rotarod, and fear conditioning tests (Supplementary Fig. 1-A, B, C). By contrast, they exhibited impairment in the object location test (Fig. 1B), suggesting that spatial learning is specifically impaired in mice with a heterozygous deletion of RP58. None of the mice used in the object location test showed extreme decreases or increases in their behavior levels of distance travelled (Supplemental Fig. 1-D). We next investigated the age-related course of this impairment. Mice at 2-, 4–5, and 12–18-months of age were used to represent adolescence (young), adulthood, and old age, respectively, in the object location test. Wild-type mice at 2 months of age preferred a familiar object in a novel location to one at a familiar location, as demonstrated by a discrimination index with values that were significantly different from zero. Though the discrimination index in 4–5-month-old mice was similar to that in mice at 2 months of age, the discrimination index in wild-type mice at 12-18-months of age was close to zero, which means impairment of object location memory (Fig. 1B). Rp58 hetero-KO mice exhibited the same level of object location memory as wild-type mice at 2-months of age. However, interestingly, at 4-5-months of age, they had a discrimination index close to zero, indicating deficits in memory. The cognitive dysfunction during aging in wild-type mice was observed at 12–18 months of age, which is old age; therefore, it is thought that reduced expression of Rp58 induced early-onset cognitive impairment in Rp58 hetero-KO mice. On the other hand, Rp58 hetero-KO mice at 4–5 months of age did not show significant behavioral effects in other behavioral tests, suggesting that spatial learning is specifically impaired in mice with a heterozygous deletion of RP58. The hippocampal CA1 region is crucial for spatial learning and theta power selectively reflects the successful encoding of new information (29). Therefore, to determine the cause of the early-onset deficit in spatial memory in Rp58 hetero-KO mice, we recorded the theta power in the CA1 region of the hippocampus (Fig. 1C). In 4-5-month-old wild-type mice, the average theta power level increased during the two seconds before and after the time of contact on day two, that is, when the objects were introduced to the animals for the first time. However, this induction in theta power was significantly attenuated on day two in 4-5-month-old Rp58 hetero-KO mice. This decrease in theta power in Rp58 hetero-KO mice was observed not only on day three but also on day two, indicating that Rp58 hetero-KO mice had a defect in learning the location of the object rather than in memory recall.

Rp58 heterozygous-KO mice show early accumulation of DNA damage in mossy cells of the dentate gyrus

Spatial learning is dependent on hippocampal function. To identify the cells in the hippocampus that contributed to the deficits in spatial memory of Rp58 hetero-KO mice, we examined the localization of RP58 protein in the hippocampus. RP58 was expressed in CA1-CA3 pyramidal cells and dentate gyrus granule cells, which is consistent with previous work (15). In the hilus of the dentate gyrus, RP58 was highly expressed in GluR2 (glutamate ionotropic receptor AMPA type subunit 2, a mossy cell-specific marker (30))-positive cells identified as mossy cells, but not in PV-positive GABAergic neurons (Fig. 2A). Thus, the present study demonstrates that RP58 is expressed not only in CA1-3 pyramidal neurons of the hippocampus and granule cells of the granular layer, but also in mossy cells of the hilus region. The expression of RP58 in these neurons prompted us to investigate the possibility that early cognitive decline is caused by the early onset of impairment in these neurons of Rp58 hetero-KO mice. RP58 may protect against DNA damage and thereby prevent cell death and contribute to cell survival (8); therefore, we examined the accumulation of DNA damage. We investigated for the presence of ssDNA in these neurons because ssDNA is generated in response to DNA damage. Interestingly, we detected ssDNA immunoreactivity in the nuclei and cytosol of many mossy cells. DNA normally resides in the nucleus and mitochondria; hence, its presence in the cytoplasm serves as a danger-associated molecular pattern (31). Mossy cells play a crucial role in spatial memory by receiving convergent excitation from granule cells, and returning bilateral, widespread, and divergent excitatory outputs to granule cells (32, 33). In wild-type mice, the number of ssDNA-positive neurons gradually increased from 2-months of age to 12-18-months of age (Fig. 2B). In Rp58 hetero-KO mice, the same level of ssDNA accumulation was detected even at 4-5-months of age as in wild-type mice at 12–18 months of age, while no significant effects were observed in 2-month-old mice compared to wild-type mice of same age. This indicated that Rp58 hetero-KO mice showed early onset of DNA damage accumulation in mossy cells. The early accumulation of DNA damage in the mutant mice was further confirmed with another marker of DNA damage, gamma-H2AX, a form of H2AX protein phosphorylated at Ser139 (34) (Fig. 2C). Cytoplasmic H2AX, presumably phosphorylated H2AX, may play an important role in cell death upon DNA damage (35). Therefore, it is likely that Rp58 plays a key role in preventing the accumulation of DNA damage in mossy cells of the dentate gyrus in the adulthood hippocampus, which is crucial for spatial learning.

Increment of activated microglia and chronic inflammation in Rp58 heterozygous-KO mice

We subsequently focused on microglia in the hilar region of the dentate gyrus because the accumulation of DNA damage in neurons is associated with activated microglia in the periphery of damaged neurons (36). In the present study, we examined microglia states in the hilus of Rp58 hetero-KO mice. In wild-type mice, the number of activated microglial cells immunostained with Iba1 and CD68 gradually increased between the ages of 2 and 12–18 months (Fig. 2D). In Rp58 hetero-KO mice, many activated microglia were observed even in 4-5-month-old mice. However, no significant activation of microglia was observed in 2- month-old mice compared with wild-type mice of the same age. The early increment of activated microglia in Rp58 hetero-KO mice raised the possibility that chronic inflammation in the brain was induced by insufficient expression of RP58 even during the early-life stage. To test this possibility, we examined gene expression profiles in the hippocampus of Rp58 hetero-KO in adulthood (4-5-months-old) and control wild-type mice in old age (12-18-months-old). Among the genes whose expression level was down-regulated in the aged wild-type mice compared with 4-5-month-old wild-type mice, 341 were down-regulated in 4-5-month-old Rp58 hetero-KO mice compared with 4-5-month-old wild-type mice. Conversely, the expression of 103 genes was up-regulated in both 4-5-month-old Rp58 hetero-KO and in aged wild-type mice compared with 4-5-month-old wild-type mice (Supplemental Fig. 2A). Pathway enrichment analysis revealed that these 103 up-regulated genes included Cxcl10, Oas2, Oas1a, and Oasl2 (Supplemental Fig. 2B), which are associated with a pathway of immune response interferon gamma action on extracellular matrix and cell differentiation (Supplemental Fig. 2C).

Ultrastructural changes in the mitochondria of Rp58 hetero-KO mice

To confirm the impaired phenotype of Rp58 hetero-KO mice, we next focused on the function of mitochondria. Mitochondria have a crucial role in antigen presentation and innate immune reactions and serve as “immunometabolic hubs” (37, 38). Abnormalities in mitochondria have been shown to be age-related (39). In the current study, we categorized the mitochondrial structure into four types, from Type I (normal) to Type IV (abnormal ultrastructure) (see Methods section for details). In 4–5-month-old Rp58 hetero-KO mice, the majority of mitochondria in neurons in the granule cell layer and in the hilar region were categorized as Types III and IV. However, in wild-type mice, the majority of mitochondria were categorized as Types I to III (Fig. 2E). It should be emphasized that the abnormal type of mitochondria categorized as Type IV, which showed i) swollen and deficient cristae or ii) swollen cristae and a discontinuous outer membrane, were detected only in Rp58 hetero-KO mice (Fig. 2E).

DNA repair is impaired in Rp58 hetero-KO mice

We aimed to develop an effective therapeutic strategy for RP58 haploinsufficiency-associated cognitive decline. We focused on the process of DNA repair based on a predicted molecular mechanism of abnormalities in Rp58 hetero-KO mice. DNA is continually damaged by exogenous and endogenous sources, but this has only minor consequences for the normal state of DNA repair. By contrast, impairment of the DNA repair pathway causes the accumulation of DNA damage, which has a harmful effect on adult health and promotes the onset of pathology (40, 41). Given that Rp58 hetero-KO mice showed early accumulation of DNA damage, we monitored the recovery of these animals from irradiation-induced DNA damage to determine whether Rp58 hetero-KO mice exhibit any defects in the DNA repair pathway. One hour after 2-month-old wild-type mice were irradiated with a single dose of 0.3 Gy (Fig. 3A), ssDNA-positive neurons were detected in the granule cell layer of the dentate gyrus, but they were not detected in non-irradiated wild-type mice (Fig. 3B). The number of ssDNA-positive neurons gradually decreased between 1–24 h after irradiation in wild-type mice (Fig. 3B). In Rp58 hetero-KO mice, a similar extent of ssDNA accumulation was observed 1 h after irradiation; however, ssDNA accumulation was still detected at 24 h after irradiation. The residual DNA damage in Rp58 hetero-KO mice, even at 24 h after irradiation, was confirmed using another marker of DNA damage, gamma-H2AX (Fig. 3C). Moreover, the ratio of activated microglia cells in the granule cell layer at 1hr after irradiation remained unchanged in Rp58 hetero-KO mice, even at 24 h after irradiation (Fig. 3D). These data support our idea that RP58 plays a pivotal role in DNA repair and microglial activation. We thus suggeted that Rp58 hetero-KO mice show early accumulation of DNA damage during aging (Fig. 2) and that this is caused by a defect in the DNA repair system (Fig. 3).

Chronic treatment with minocycline prevents early-impaired aging phenotypes in Rp58 hetero-KO mice

We aimed to develop an effective therapeutic strategy for cognitive impairment by identifying agents that attenuate the early onset of cognitive dysfunction in RP58 hetero-KO mice. Thus, we investigated the effect of minocycline, which has neuroprotective (19) and anti-inflammatory effects (18), on the phenotypes of Rp58 hetero-KO mice. Rp58 hetero-KO- and control mice were administered minocycline for 1- to 4–5 months in their drinking water (Fig. 4A). Chronic treatment with minocycline prevented the increase of ssDNA-positive and/or gamma-H2AX-positive neurons in the dentate gyrus of 4-5-month-old Rp58 hetero-KO mice (Fig. 4B, 4C). Furthermore, we examined the effect of minocycline on the early increment of activated microglia in Rp58 hetero-KO mice. Chronic treatment with minocycline prevented the increment of activated microglia in the dentate gyrus of Rp58 hetero-KO mice (Fig. 4D). Our findings indicate that minocycline is effective in preventing a series of early-impaired functions, namely DNA damage accumulation and an increment of activated microglia, caused by the under-expression of RP58 in the dentate gyrus. Since it is important to assess the effects of any treatment strategy on behavior, we also examined the effect of minocycline on the impairment of object location memory in 4-5-month-old Rp58 hetero-KO mice (Fig. 4E). Minocycline prevented cognitive dysfunction in Rp58 hetero-KO mice but did not affect cognitive function in wild-type mice. Taken together, our results showed that Rp58 is involved in inflammation and in the repair of DNA damage and that its reduced expression leads to impaired cognitive function. These findings suggest that minocycline administration could have potential as a therapeutic strategy for the prevention of the cognitive impairment associated with RP58 haploinsufficiency via its ability to prevent DNA damage accumulation and an increment of activated microglia.

In this study, we analyzed Rp58 hetero-KO mice, which we established as a model of ID caused by ZBTB18/RP58 haploinsufficiency, to obtain insights into the pathogenesis of ZBTB18/RP58 haploinsufficiency in adulthood and identify preventive treatment methods. Rp58 hetero-KO mice showed impaired spatial cognition, accumulation of DNA damage in neurons in the hippocampal dentate gyrus hilus, as well as an increment of activated microglia in adulthood. In the hippocampal dentate gyrus hilus, RP58 was strongly expressed in mossy cells but not in PV-positive cell GABAergic neurons, suggesting that heterozygous loss of RP58 induced an abnormality of mossy cells in adulthood. Physiological analysis revealed that the regular increase in theta power in the hippocampal CA1 region during the learning period is attenuated during adulthood in Rp58 hetero-KO mice; theta power in the CA1 region is involved in dentate granule cell activity, which is regulated by mossy cell circuitry (42). Therefore, the dysfunction of mossy cells is probably contribute to the impairment of theta power in the CA1 region of Rp58 hereto-KO mice. In other words, it is possible that the impairment of mossy cells in Rp58 hetero-KO mice impairs spatial cognition because mossy cells play a crucial role in spatial memory. Mice with the RP58 heterozygous deletion showed accumulation of DNA damage in a group of neurons in the hippocampal dentate gyrus hilus, suggesting that RP58 may be involved in DNA damage repair. Thus, we performed artificial DNA damage experiments in the present study. The results showed that the hippocampal dentate gyrus neurons of RP58 hetero-KO mice were less proficient in DNA repair than those of wild-type mice, suggesting that RP58 is required for DNA repair.

The present study showed that minocycline inhibited microglial polarization to a proinflammatory state in Rp58 hetero-KO mice. Minocycline selectively inhibits activation of injurious microglia (18, 43). Microglia, the resident brain glial population most associated with neuron protection in health and their destruction in disease, could be a significant player in age-related changes. With aging, microglia change from a ramified morphological state to a de-ramified spheroid structure with abnormal processes (44). In the present study, an increment of activated microglia was detected in 12-18-month-old wild-type mice. By contrast, an increment of activated microglia was present even in 4-5-month-old Rp58 hetero-KO mice, but not in wild-type mice of the same age. Microglia protect neurons via inflammatory responses and phagocytosis in the early phase of activation by exogeneous or endogenous disorders, but they have detrimental effects in the late phase. In the present study, activated microglia in the late phase may deteriorate the state of neurons in the hippocampal dentate gyrus hilus. Recently, it is demonstrated that RP58 in hepatocyte regulates NLRP3 inflammasome’s activity in macrophage and inflammation in liver (45). Relying on this, RP58 haploinsufficient mossy cells may activate microglia via the NLRP3 inflammasome, inducing neuroinflammation and injurious of neurons. In this case, minocycline is considered to have an effect in preventing an increment of activated microglia via inhibition of NLRP3 inflammasome (43, 46–49), which may inhibit an exacerbation of the state of surrounding neuron. The hypothesis postulates that accumulation of DNA damage is caused by increment of activated microglia in late phase. However, it is unclear by what mechanism DNA damage accumulates in the neurons of RP58 heterozygous mice. On the other hand, the accumulation of DNA damage in mutant mice was suppressed by pretreatment with minocycline. Minocycline treatment is known to induce autophagy via FOXO (50), which is cooperate with RP58 in a regulation of function (51). RP58 is a transcription factor that represses transcription of a series of genes whose expression represses autophagy (52) and decreases in the aging brain (53). The decrease in autophagy with age is thought to be one of the causes of the accumulation of DNA damage (54). Therefore, the reduction of RP58 may reduce autophagy capacity, which lead to the accumulation of DNA damage in mossy cells. Injured mossy cells with accumulated DNA damage by RP58 haploinsufficiency released injurious substances, e.g., damage-associated molecular patterns(DAMPs), which may activate microglia via NLRP3 inflammasome (17). Therefore, it cannot be ruled out the possibility that minocycline affects autophagy pathway controlled by RP58. Further studies are needed to elucidate the mechanism by which RP58 regulates DNA damage repair. These observations suggested that minocycline might have potential as an agent for the treatment of patients with ZBTB18/RP58 haploinsufficiency. Minocycline is already used as a treatment for inflammatory diseases, and clinically, drug repositioning is easier than drug development. Therefore, the present findings demonstrate that minocycline could have potential for the prevention of cognitive decline in patients with RP58 haploinsufficiency.

At 2 months of age, the young adult stage, the hippocampal dentate gyrus of Rp58 hetero-KO mice is healthy and has adequate spatial cognitive function, similar to that of wild-type mice. At 12–18 months of age, the aging stage, DNA damage accumulation and an increment of activated microglia were observed in the hippocampal dentate gyrus of Rp58 hetero-KO and wild-type mice, and both mice showed defects in cognitive function. By contrast, at 4–5 months of age, the early aging stage, the Rp58 hetero-KO mice showed impairment of the hippocampal dentate gyrus and reduced spatial cognition, similar to those observed in the aging stage, while wild-type mice had no impairments in these functions and were identical to younger wild-type mice. Rp58 hetero-KO mice had the same level of cognitive function as wild-type mice at 2 months, but not at 4–5 months, suggesting that the 4–5-month-old mice may have mimicked dementia, an acquired cognitive impairment, as well as ID, an inherent cognitive impairment. Dementia is an established comorbidity in persons with ID (55), and ID itself is an established risk factor for dementia (56). Although the symptoms of ID may appear to be asymptomatic, their effects may become apparent with age. RP58 expression is markedly reduced in the cerebral cortex of elderly humans (57). We might trace change of cognitive function in ID over a lifetime using Rp58 hetero-KO mice. Early cognitive decline in Rp58 hetero-KO mice was associated with impaired DNA repair, and many early aging syndromes, such as Werner syndrome, are known to be caused by mutations in DNA repair-related genes (58). In the present study, we reported mitochondrial defect in RP58 hetero-KO mice in adulthood. Nuclear DNA damage is known to spill over into mitochondria, triggering mitochondrial damage and furthermore, this increases with aging (59). These findings are consistent with the observation that reduced expression of RP58 induces cognitive decline in adulthood, i.e., dementia. Thus, RP58 hetero-KO mouse may have accelerated age-related functional decline, and could be used as a model animal to analyze the relationship between ID and dementia, which may be useful for analyzing the molecular mechanisms of brain development and aging.

Four to 5 month-old Rp58 hetero-KO mice did not show significant behavioral effects in the open field, rotarod, and fear conditioning tests, suggesting that mice with a heterozygous deletion Rp58 do not show all of the features of age-associated brain dysfunction. Considering their accelerated age-related functional decline, it should be noted that RP58 hetero-KO mice may exhibit other behavioral abnormalities in addition to that in object location memory at different periods. Since our findings suggest that heterozygous loss of RP58 results in the accumulation of DNA damage, immune system dysfunction, and early decline in cognitive function, we suggest that RP58-mediated DNA repair pathways and immune system function may be important for the development and maintenance of cognitive function.

Availability of data and materials

The datasets used and/or analyzed in the current study are available from the corresponding author on reasonable request.

Funding

This work was partially supported by JSPS KAKENHI (18H02537 to HO, 22H02729 to HO, 23K18169 to HO, 17K16408 to TT, 21H05130 to HY, and 21K18231 to HY), by AMED-PRIME program (20gm6110010h0004 to HY), by AMED-FORCE Program (22gm4010019h0001 to HY), by JST-FOREST Program (JPMJFR2150 to HY), by Takeda Science Foundation to SH and by a Research Grant for Public Health Science, The Naito Foundation and Suzuken Memorial Foundation awarded to TT.

Acknowledgments

The authors thank Dr. Masataka Kasai for the use of RP58 mutant mice, Dr. Minoru Saitoe, Dr. Nobuhiro Kurabayashi, Dr. Makoto Hashimoto, and Dr. Hideki Miwa for advice on the work presented here, and Kazuki Shiotani and Yuta Tanisumi for their help with the analysis of the electrophysiology data. We would also like to thank Bioedit Ltd (www.bioedit.com) for English language editing.

Author contributions

T.T., S.H. and H.O. designed the research. H.Y. and H.O. supervised the study. T.T. performed and analyzed most experiments. K.E. performed the sample preparation for electron microscopy. Y.N. performed the complementary DNA microarray analysis of gene expression. H.M. assisted with the setup for the electrophysiological experiment. H.S. performed a part of immunostaining and provided additional help with this study and made important suggestions. T.T. generated all figures and tables. T.T. and H.Y. wrote the manuscript. H.O. edited the manuscript.

Ethics approval

All experimental procedures were approved by the Animal Experimentation Ethics Committee of the Tokyo Metropolitan Institute of Medical Science (49040).

Consent for publication

Not applicable.

Declaration of interest

The authors declare no competing interest.

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No competing interests reported.

Keyresourcetable.xlsx
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Supplemental fig. 1 Analysis of other behaviors in Rp58 hetero-KOmice (A) Horizontal activity and time in center were calculated using an open field test. Horizontal activity was calculated per 10 min. Two-way repeated ANOVA followed by Holm’s test and paired t-test vs. age matched wild-type mice; n = 14 for 4–5-month-old wild-type mice, n = 11 for 4–5-month-old Rp58 hetero-KO mice, (B) Motor learning function was calculated using the Rota-rod test. The latency to fall was recorded for five consecutive days. Two-way repeated ANOVA followed by Holm’s test; n = 12 for 4–5-month-old wild-type mice, n = 11 for 4–5 month-old Rp58 hetero-KO mice, (C) Contextual fear memory was calculated using the fear-conditioning test. The freezing time was calculated per min. Two-way repeated ANOVA followed by Holm’s test; n = 23 for 4–5-month-old wild-type mice, n = 18 for 4–5 month-old Rp58 hetero-KOmice, (D) Data from the object location test were obtained. Total horizontal activities were calculated on day 3. Paired t-test vs. age matched wild-type mic; n = 12 for both genotypes at 2- or 4–5-months of age.
sup2.eps
Supplemental fig. 2 (A) Venn diagrams depicting an overlap in hippocampus genes exhibiting a < 0.5-fold or >1.5-fold change in expression in 12–18-month-old wild-type mice compared with 4–5-month-old wild-type mice. n = 3 per group. (B) Scatter plot depicting 103 up-regulated genes. (C) Top 4 pathway enrichment analysis of commonly expressed genes.

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published 12 Oct, 2024

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Minocycline prevents early age-related cognitive decline in a mouse model of intellectual disability caused by ZBTB18/RP58 haploinsufficiency

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Abstract

Figures

Introduction

Materials and Methods

Experimental model

Animals

Behavioral assessments

Electrophysiology

Analysis of electrophysiology data

Immunohistochemistry

Analysis of immunostaining

Irradiation and immunohistochemistry

Electron microscopy

Drug treatment

Transcriptome analysis

Statistical analysis

Results

Rp58 heterozygous-KO mice exhibit early-onset cognitive decline

Increment of activated microglia and chronic inflammation in Rp58 heterozygous-KO mice

Ultrastructural changes in the mitochondria of Rp58 hetero-KO mice

DNA repair is impaired in Rp58 hetero-KO mice

Chronic treatment with minocycline prevents early-impaired aging phenotypes in Rp58 hetero-KO mice

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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