A mouse model of sporadic Alzheimer’s disease with elements of major depression

doi:10.21203/rs.3.rs-3781115/v1

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A mouse model of sporadic Alzheimer’s disease with elements of major depression

https://doi.org/10.21203/rs.3.rs-3781115/v1

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Animals after bulbectomy are often used as a model of major depression or sporadicAlzheimer’s disease and, hence, the status of this model is still disputable. To elucidate the nature of alterations in the expression of the genome after the operation we analyzed transcriptomes (RNA-seq data) of the cortex, hippocampus, and cerebellum of olfactory bulbectomized (OBX) mice. Analysis of the functional significance of genes in the brain of OBX mice indicates that the balance of the GABA/glutamatergic systems is disturbed with hyperactivation of the latter in the hippocampus leading to the development of excitotoxicity and induction of apoptosis on the background of severe mitochondrial dysfunction and astrogliosis. On top of this, the synthesis of neurotrophic factors decreases leading to the disruption of the cytoskeleton of neurons, an increase in the level of intracellular calcium, and activation of tau protein hyperphosphorylation and beta-amyloid depositions. Moreover, the acetylcholinergic system is deficient in the background of hyperactivation of acetylcholinesterase. Importantly, the activity of the dopaminergic, endorphin, and opiate systems in OBX mice decreases leading to hormonal dysfunction. Genes responsible for the regulation of circadian rhythms, cell migration, and impaired innate immunity are activated in OBX animals. All this takes place on the background of drastic down-regulation of ribosomal protein genes in the brain. The obtained results indicate that OBX mice represent a model of Alzheimer's disease with elements of major depression. This model can be tentatively attributed to AD subtype B2 in humans.

bulbectomized mice

Alzheimer's disease

depression

transcriptomic analysis

Alzheimer's disease (AD) is the most common form of dementia among older patients and one of the leading causes of their death. Currently, there is an active search for new treatment of AD, since effective methods to cure this disease have not yet been found due to the complex etiology and multifactorial nature of AD, the genesis of which involves both genetic disorders and the influence of adverse environmental factors [1].

Recently, analysis of transcriptomic data from AD patients, as well as several transgenic animal models of AD, has made it possible to identify at least three major subtypes of AD with different pathophysiological mechanisms. These mechanisms include combinations of dysregulation associated with sensitivity to tau-mediated neurodegeneration, amyloid formation and neuroinflammation, synaptic signaling, immune status, mitochondrial organization and myelination [2]. Subtypes of AD vary in sensitivity to different drugs, which makes it important to determine them for targeted therapeutic intervention. Although there are currently more than a hundred transgenic models of AD of the first, second, and third generations [3], experience has shown a very limited possibility of translating the data obtained from their studies to the clinic. Therefore, modeling different subtypes of the most common sporadic AD is necessary both to study the mechanisms of pathology and to develop effective treatment.

In contrast to numerous transgenic models of AD [4–6], there is a very limited number of rodent models of sporadic AD. One such model is represented by various rodents with removed olfactory bulbs [7].

The idea of a relationship between AD and a decrease in olfactory sensitivity was first proposed by Ferreyra-Moyano and Barragan [8], who demonstrated that deterioration of the sense of smell is often observed at the early stages of the AD. Changes in olfactory sensitivity occur in 90% of patients with this pathology and can be used to diagnose this disease [9, 10].

It is also known that patients with AD exhibit morphological changes in the cortical and subcortical olfactory structures connected with the olfactory bulbs, where generation of numerous senile plaques containing beta-amyloid protein, neurofibrillary tangles and death of mitral cells are noted [11]. However, the question remains unresolved: either olfactory disorders in AD represent a consequence of pathology developing in the brain, or they can act as an initiating factor of the pathology [12].

A model for studying the consequences of olfactory impairment is represented by animals with removed olfactory bulbs, the so-called “olfactory bulbectomized” (OBX) mice and rats. OBX animals were characterized by a pronounced impairment of spatial memory, correlated with a progressive neurodegenerative process in brain structures such as cortex and hippocampus also observed in patients with AD. Characteristically, in OBX rodents, the volume of the hippocampus decreases [13], the functioning of synapses and mitochondrial metabolism is disrupted [14], and increased cell death is observed [15, 16]. All this happens on the background of severe dysfunction of the acetylcholinergic [17], and serotonergic brain systems [18].

In extracts of the neocortex and hippocampus of OBX animals, an increased level of the soluble β-amyloid peptide is detected [19]. Interestingly, in OBX guinea pigs, which have a primary Aβ structure similar to the structure of human Aβ, typical Aβ plaques are formed in the cortex and hippocampus [20]. Moreover, ablation of the olfactory bulbs in B6C3Tg (APPswe, PSEN1dE9) 85Dbo/J transgenic mice significantly accelerated the deposition of amyloid plaques in the brain [7].

Mice knockout for the cyclic nucleotide-gated channel alpha 2 (Cnga2) gene, which is important for the transduction of olfactory signals, also exhibited signs of AD, including impaired learning and memory, loss of dendrite spines, as well as decrement of synaptic proteins [21]. Experiments on live brain slices from OBX rats revealed a significant decrease in long-term potentiation on synapses of Schaffer collaterals in neurons in the CA1 field of the hippocampus [22, 23] and a decrease in the level of synaptophysin in the temporal cortex and hippocampus, indicating disorders of synaptic plasticity. This animal model also showed a decrease in mushroom-type spines and tau hyperphosphorylation [24].

It should be noted that drugs approved for the treatment of AD, in particular cholinesterase inhibitors and the NMDA receptor antagonist memantine, is able to alleviate cognitive impairment in OBX mice [25]. We have also previously showed that OBX mice respond similarly to therapeutic intranasal administration of recombinant human HSP70 as the classical transgenic model of AD, the 5XFAD mice [26]. All of the above facts indicate the validity of using OBX animals as a model of the sporadic AD.

An important advantage of the OBX model is its speed (symptoms are evident one month after bulbectomy) and high reproducibility, which distinguishes it from most other models of sporadic and familial AD. It should be noted, however, that OBX animals is currently more often used as a model of endogenous depression (major depression - MD) [27], which is believed to develop as a result of deprivation of basic for rodents odor information about the environment. OBX mice exhibit a few behavioral and morphological changes that are also characteristic of this pathology, which has become a source of controversy and dispute as to which disease OBX animals are a model of [11, 28].

Indeed, in OBX rodents, increased locomotor activity, aggressiveness, and increased sensitivity to stress are observed, along with deficits in passive avoidance and hyperactivity in the open field test, that are normalized by antidepressants [29].

OBX surgery leads to a decrease in the concentrations of norepinephrine, serotonin, and 5-hydroxy-indoleacetic acid in the amygdala, frontal cortex, and midbrain [30].

Furthermore, compensatory mechanisms ensure an increase in the level of β-adrenergic receptors on blood lymphocytes and in the amygdala cortex, as well as 5HT2 receptors in the neocortex [18], that are characteristic symptoms of patients suffering from endogenous depression. Interestingly, caffeine has a therapeutic effect in OBX animals [31]. Based on the above, we may conclude that OBX mice are a unique and very convenient model for studying the mechanisms underlying AD-MD comorbidity.

Herein, we report the results of transcriptomic analysis of tissues of three brain structures (the cortex, hippocampus, and cerebellum) of OBX mice in comparison with sham-operated (SO) mice. We evaluated the enrichment in gene sets and compared them with the data obtained from various public resources containing functional and disease information for MD and different subtypes of AD. This analysis revealed significant changes in gene expression in the studied brain regions of OBX mice characteristic for AD and major depression which enable to conclude that this model belongs to a new subtype of AD, characterized by a few manifestations of MD.

2.1. Animals

Male NMRI (22–24 g) mice were purchased from Beijing HFK Bioscience Co., LTD (Beijing, China). Animals were maintained under conditions of standard lighting (light on at 08:00–20:00), temperature (23- 25⁰C), and humidity (55%) with free access to food and water. All animal experiments were conducted in agreement with the Provision and General Recommendation of Chinese Experimental Animals Administration Legislation and were approved by the Animal Ethics Committee in Institute (SLXD-20180912006, 12 September 2018).

2.2. OBX Surgery

After a seven-day adaptation, mice were subjected to bilateral OBX according to the previously described methods [26]. Mice were anesthetized with 2% pentobarbital sodium (40 mg/kg, intraperitoneal injection (i.p.), and fixed in stereotaxic apparatus. After the skull was exposed, one hole was drilled on the midline (2 mm in diameter, 3 mm anterior to bregma). The removal of the olfactory bulbs was performed by aspiration with a blunt syringe needle attached to a vacuum pump. The hole was filled with an absorbable collagen sponge immediately to control the bleeding. Sham-operated mice (SO) experienced the same procedures, but their bulbs were left intact. After surgery, the mice were housed in an individual cage and given penicillin sodium (10,000 U in normal saline, i.m.) once a day to prevent probable infection.

All procedures were conducted by national and EU (Directive 2010-63EU) guidelines regulating animal research and were approved by the local ethics committee (CEEA-PRBB).

2.3. RNA Isolation, Library Preparation, and Transcriptome Sequencing

For the transcriptomic analysis, three brain tissues (the hippocampus, cortex, and cerebellum) from two groups (OBX and sham-operated) were used. Brains were dissected and stored at 80⁰C until the RNA isolation.

The extraction RNA was made using RNAzol RT (Molecular Research Center, Cincinnati, OH, USA) according to the company’s protocol. The concentration and quality of RNA were determined via a Qubit Fluorometer (Invitrogen) and an Agilent BioAnalyzer 2100, respectively, using an RNA 6000 nano kit (Agilent Technologies, Santa-Clara, CA, USA). For libraries RNA with Integrity Number (RIN), no less than 8 were taken. Illumina NEB Next Ultra II Directional RNA Library Prep Kit (NEB, Ipswich, MA, USA) was used for mRNA library preparation. The sequencing was performed on the Illumina NextSeq 2000 platform. RNA sequencing and further differential expression estimation were performed using the equipment of the Engelhardt Institute of Molecular Biology RAS “Genome” center (http://www.eimb.ru/rus/ckp/ccu_genome_c.php, accessed on 22 February 2022). The sequencing data obtained in the present work are available at the NCBI Sequence Read Archive (project ID GSE249297 GEO base).

2.4. RNA data processing

As a result of deep RNA sequencing, we obtained 10–20 mln 75-bp single-end reads per one biological sample. Raw RNA-seq reads processing was performed using the PPLine package [32]. We used Adaptor sequences that were trimmed off, and low-quality reads were removed using Cutadapt [33]. Reads were aligned to the reference genome (GRCm39 v.104) using STAR aligner [34] with total alignment rate > 95%. SAM file post-processing was performed using the Samtools software package [35]. Reads, assigned to exons of protein-coding genes were counted using feature Counts utility from the subread package [36]. Subsequent analysis of differential expression was performed in R language version 4.2.2, using RTrans pipeline. Raw gene expression counts were normalized using the TMM method implemented in the edgeR package [37], and, subsequently, each gene was tested on differential expression using the quasi-likelihood negative binomial generalized log-linear model. Genes with p-value < 0.05 were considered as differentially expressed.

Gene set enrichment analysis (GSEA) was performed using the clusterProfiler package [38] with GeneOntology [39] and KEGG [40] databases. Heatmaps and Venn diagrams were plotted using ComplexHeatmap and VennDiagram R packages.

Genes, representing key markers of Alzheimer’s disease [2, 41, 42], major depression disorder [43], apoptosis (KEGG pathway mmu04210), ferroptosis [44] and metabolism of mitochondria [45] were downloaded from open-access published data.

To estimate a resemblance of gene expression patterns between OBX mice and human subtypes of Alzheimer’s disease, we used LogFC values published in open-access data [46]. Pairs of orthologous genes among mouse and human genomes were matched using OrthoDB database v.11 [47]. Arrays of LogFC values were compared using Spearman’s coefficient of correlation.

Protein-protein interaction networks of products of differentially-expressed genes were created in StringDB v.11 [48] with parameters “physical interaction network” and “confidence level = 0.7”. Non-interacting nodes were removed. The network was clustered using k-means (mcl method) with several clusters k = 5, each cluster was tested separately on representative sets of enriched biological processes with the clusterProfiler package [38]. Visualization of the interaction network was performed using Cytoscape software.

1. RNA sequencing and analysis of differentially expressed mRNAs in three brain structures of OBX mice.

We performed the comparative analysis of the transcriptome signatures of three brain structures of OBX and control SO 4-month-old mice, one month after bulbectomy when major impairments of spatial memory are already developed [49]. We determined the significant differentially expressed (DE) genes with adjusted p-value < 0.05, deregulated in the three brain areas indicated above. The removal of the olfactory bulbs had a strong effect on gene expression in all brain areas studied. Among the total genes expressed in the hippocampus of the OBX mice, there were 944 up-regulated and 1036 down-regulated genes as compared with control SO animals. In the сortex and cerebellum, 879 and 557 genes are expressed correspondently, of which 487 up-regulated and 392 down-regulated genes in the cortex while 277 up-regulated and 280 down-regulated genes were revealed in the cerebellum (Fig.1 A, B, C).

The comparative analysis did not reveal significant differences between the number of up and down-regulated genes in each of the studied brain areas in OBX mice (Figure 1C). However, the hippocampus was significantly superior to other brain areas in terms of the total number of genes with changed expression in OBX mice in comparison to SO animals (Figure 1).

The results of transcriptome analysis were corroborated by the RT-PCR technique in all three brain areas. Four genes differentially expressed in OBX mice i.e., Apba1, Atn1, Kmt2d, and Ttbk1 were used for PCR analysis. (Suppl. Figure S1).

2. Functional annotation of genome expression changes in OBX mice

The olfactory bulbectomy led to drastic changes in gene expression, resulting in the activation of several signaling pathways. Fig 2 depicts the top GO enrichment domains of the target genes with the significantly dysregulated mRNAs. The major affected biological processes according to GO enrichment are involved in neurotransmitter secretion, cognition, learning and memory, neuropeptide signaling pathway, metabolic processes, ribosome processing and biogenesis, cell-matrix adhesion, etc. The hippocampus represents a structure where the most drastic changes in the expression of pertinent genetic pathways took place in the OBX mice.

GSEA analysis using the GO database revealed pronounced up-regulation in many important pathways and signal systems taking place in the studied brain areas with the hippocampus being the structure where most drastic changes took place. Thus, up-regulation of genes responsible for neuronal plasticity, neuropeptide signaling pathway, neurotransmitter transport, and secretion, as well genes responsible for regulation of glucose metabolism and WNT as well as Notch signaling were significantly up-regulated in the hippocampus of OBX mice.

Similarly, the KEGG pathway analysis also revealed pronounced up-regulation in the expression of genes involved in axon guidance, GABAergic synapse, and other synapses as well as synaptic vesicle cycle, circadian entrainment, different signaling pathways, including WNT and Notch signaling in OBX-mice (Fig. S2). In all these gene categories most pronounced up-regulation also took place in the hippocampus of OBX mice and to a significantly lesser degree in the cortex and cerebellum. Characteristically, pronounced down-regulation of genes involved in the protein synthesis such as rRNA processing and ribosome biogenesis represent prominent exceptions demonstrated by using both GSEA and KEGG databases.

At the next step, we monitored the expression of genes involved in the metabolism of β-amyloid and Tau protein in the brain of OBX mice.

3. Study of gene expression in the brain regions of OBX mice related to the metabolism of beta-amyloid and tau protein

It is well known that neurodegenerative changes in the brain of AD patients are associated with the presence of oligomeric forms of amyloid beta protein and tau protein fibrils, as well as with the processes of their formation and utilization. Therefore, we analyzed the expression of known genes related to these proteins, as well as the pathways of their production and utilization in OBX mice (Figure 3).

Analysis of gene expression in three brain regions of OBX mice indicates that the most dramatic changes took place in the hippocampus. The detected genes with altered expression are related to both the formation of beta-amyloid and possible compensatory mechanisms aimed to ameliorate this process. It is noteworthy that in the hippocampus, 28 genes out of 45 showed upregulation and only 8 exhibited downregulation.

Several genes that increased their expression in the hippocampus and partially in the cortex of OBX mice are of particular interest. They are represented by the Ywhag gene encoding 14-3-3γ protein, which increased GSK-3β activation and promoted tau phosphorylation in Alzheimer's disease [50], Fn1 – fibronectin1, which declined at the onset of remyelination of the lesion area of the CNS and is implicated in BBB breakdown [51]. All studied brain structures of OBX mice were characterized by a pronounced increase in the level of expression of the Sox9 gene, which responds to Aβ deposition [52]. Increased expression in the cortex and hippocampal formation was also demonstrated by the Tau tubulin kinase-1 (Ttbk1) genes, the activity of which leads to the deposition of hyperphosphorylated tau.

Among the genes that showed decreased expression in the brain of OBX mice, the prefoldin genes Pfdn5 in the hippocampus, as well as Pfdn2 in all analyzed structures, should be mentioned. These genes normally function as chaperones. Furthermore, in the hippocampus of OBX mice, the reduced expression of the gene Prepl encoding protein PREPL and down-regulation of the neuron-specific gene, Rasgefl1c (RasGEF Domain Family Member 1C), were observed which may be involved in cytoskeletal degeneration [53], and late-onset neurocognitive disorders [54].

On the other hand, most of the genes involved in β-amyloid synthesis exhibited significant up-regulation in OBX mice. We also detected probably compensatory up-regulation of genes responsible for APP stabilization (Apba1, Apba2) and receptor-mediated activation of Syk, which should reduce Aβ load by up-regulation of microglial phagocytosis [55]. Finally, the observed up-regulation of Piezo1 and Sorla/Sorl1 genes in the brain of OBX mice is also apparently of a compensatory nature because it ameliorates brain Aβ burden [56] and can decrease the number of amyloidogenic products in the affected individuals [57]. To this end, it was shown that microglia lacking Piezo1 led to the exacerbation of Aβ pathology and cognitive decline, whereas pharmacological activation of microglial Piezo1 ameliorated brain Aβ burden and cognitive impairment in 5xFAD mice [56].

Thus, in the brain of OBX mice, the expression was significantly changed for genes that promote beta-amyloid deposition and hyperphosphorylation of tau protein characteristic of AD and for genes that may compensatory prevent the development of this neuropathology. At the next stage we analyzed genes involved in the functioning of neurotransmitter systems that changed their expression after bulbectomy in the studied brain regions.

4. Expression of genes involved in various neurotransmitter systems in the brain of OBX mice

Since the main goal of this study was to determine what pathology MD or AD represent OBX animals, we first analyzed the expression of genes, judging by the literature data, related to the functioning of neurotransmitter and several other receptor systems characteristic for AD and MD in the studied brain structures of OBX mice. Table 1 lists the genes of neurotransmitter systems that underwent the greatest changes in expression in the brain of OBX mice.

Table 1. Genes involved in the activity of the main neurotransmitter systems,

with the largest changes in expression in OBX mice compared to SO animals. Blue–down reg. genes, red–up reg. genes

System	Gene
System	Hippocampus	Cortex	Cerebellum
Glutamatergic	Grm1
	Grm4
	Slc1a1
	Slc1a2	Slc1a2
GABAergic	Gabarapl2		Gabarapl2
Cholinergic	Ache	Ache
	Chrm1
	Chrm4
	Chrna4
Dopaminergic	Arpp21
Dopaminergic	Drd2
Neurotransmitter transport	Slc6a20b		Slc6a20b
Long-term depression	Irs2	Irs2	Irs2
Neuroactive ligand-receptor interaction	Adcyap1r1	Adcyap1r1
	Adora2a
	Cckbr
		Hrh3
		Mas1
	Oprl1
	Sstr1
	Sstr4

Table 1 demonstrates that OBX mice are characterized by a deficiency of the GABAergic system since the expression of the gene gamma-aminobutyric acid (GABA) A receptor-associated protein-like 2 which mediates the fast inhibitory synaptic transmission in the central nervous system is drastically reduced. At the same time, activation of the glutamatergic system is noted in the hippocampus, which is manifested in increased expression of metabotropic glutamate receptors. Activation of the glutamatergic system may also be indicated by a compensatory increase in the expression of Slc1a1 genes encoding members of the high-affinity glutamate transporters. OBX mice were also characterized by impaired expression of the Chrm1 and Chrm4 genes, associated with the activity of the acetylcholinergic system and responsible for the synthesis of muscarinic acetylcholine receptors M1 and M4, that have long been considered to be involved in the pathophysiology of AD [58-60].

In the cortex and hippocampus of OBX mice, there is an increase in the expression of the gene encoding the alpha-4 subunit of the neuronal nicotinic acetylcholine receptor (Chrna4) [61, 62], as well as a strong increase in the expression of the acetylcholinesterase gene (Ache). Hyperactivation of this gene is associated with synaptic acetylcholine deficiency and the development of cognitive impairment in AD [63-66].

On the other hand, in the hippocampus of OBX animals, a pronounced decrease in the expression of dopamine D2 receptor genes (Drd2) is observed, as well as a decrease in the expression of the gene that regulates the effects of dopamine itself (Arpp21). In addition, down-regulation of the expression of the Slc6a20b gene, responsible for the transport of neurotransmitters, is observed in the hippocampus and cerebellum of OBX mice. Similar disturbances in the dopaminergic system, along with loss of dopaminergic neurons, as well as decreased expression of dopamine D2 receptors in the hippocampus, have been revealed in a classical mouse model of AD, and these disturbances have been associated with early manifestations of AD [67, 68].

Interestingly, OBX mice also showed changes in the expression of genes responsible for the synthesis of peptide receptor ligands, which was manifested in a decrease in the expression of somatostatin receptor genes 1 and 4 (Sstr1 and Sstr4). These results are consistent with other data enabling to suggest that the downregulation of these genes (SSTs) represents an early pathological signature of AD ([69, 70].

In the cortex of OBX mice, an increase in the expression of the histamine receptor 3 (Hrh3) gene was demonstrated. This gene is one of the targets of therapeutic agents being developed for the treatment of numerous disorders, including cognitive diseases such as attention deficit hyperactivity disorder and AD [71].

It is noteworthy that in the cortex of OBX mice, there is a decrease in the expression level of the MAS1 receptor gene (Mas1), the activation of which leads to a decrease in blood pressure. Besides, in the hippocampus and cortex an increased level of expression of the substrate of insulin receptor gene (Irs2) was noted. Deficiency of MAS receptors (Masr) and angiotensin (1-7) on the background of excess amounts of angiotensin II was often observed in sporadic AD, and its restoration has a positive effect in AD patients [72, 73].

It should be underlined that the expression of genes that determine the development of pathology does not always coincide in OBX and AD. For example, OBX animals exhibited a significant decrease in the expression of the adenosine receptor A2a (Adora2a) gene, while patients with AD, on the contrary, are characterized by increased expression of this receptor [74, 75].

Interestingly, bulbectomy also caused an increase in the expression of the nociceptin opioid peptide receptor gene (Oprl1) in the hippocampus, which is characteristic of both depression and AD [76].

From the data presented in Table 1, it is clear that the bulbectomy causes serious changes in the expression of genes of receptor systems. These changes manifest in an imbalance of the inhibitory and excitatory systems in the brain of OBX mice, and also lead to a deficiency of the dopaminergic and acetylcholinergic systems. The observed changes in the activity of neurotransmitter systems are probably responsible for the characteristic behavioral changes observed in OBX animals.

3. Mitochondrial genes differentially expressed in OBX mice.

At the next stage, we analyzed differentially expressed genes associated with the functional state of mitochondria in OBX mice using the MitoCarta 3.0 database.

From the top 50 genes related to the state of mitochondria, 36 genes decreased their expression in the brain of OBX mice compared to SO animals. It is necessary to emphasize the drastic decrease in the expression of genes belonging to Mrp and Nduf families. The activity of these genes is associated with the synthesis of mitochondrial ribosomal proteins and subunits of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I) which plays a vital role in cellular ATP production, the primary source of energy for many crucial processes in living cell. It is known that mitochondrial Complex I deficiency causes adult-onset of several neurodegenerative disorders [78].

In OBX mice the expression of the Fmc1 gene, encoding assembly factor 1, responsible for the formation of mitochondrial Complex V, is significantly reduced in all three studied brain areas. Pronounced down-regulation was also noted for the Pet117 gene. Depletion of Pet117 reduced mitochondrial oxygen consumption rate and impaired mitochondrial function. This gene plays a role in the biogenesis of mitochondrial complex IV or cytochrome c oxidase, which is part of the respiratory electron transport chain of mitochondria [79]. Furthermore, the decrease in Mgst gene expression observed in the brain of OBX mice should reduce the protection of the outer mitochondrial membrane from oxidative stress [80].

Only 14 genes out of 50 depicted in Figure 4 exhibited moderate, apparently compensatory activation of expression, including the Mief1 and Slc25a51 genes. These genes help to maintain energy metabolism in the cell [81] and improve mitochondrial respiration [82]. There is also activation of genes encoding the GPT2 proteins in the hippocampus and cortex, as well as Mcu in the cortex, that are responsible for the regulation of cell growth, amino acid metabolism, and level mitochondrial calcium. An increase in their expression also apparently indicates the compensatory nature of these changes.

Increased expression of the Kmt2d gene [83] observed in all three brain areas, and up-regulation of three other genes in hippocampus (Mlxip, [84], Wdfy4 [85], and Lamb2 [86], whose activity is associated with the suppression of apoptosis and stabilization of synapses (Figure 4) are probably also represent compensatory reaction. However, given the deficiency of mitochondrial ribosomal proteins found in OBX, even increased expression of genes aimed at compensating for severe mitochondrial dysfunction is unlikely to have a significant effect.

Next, we analyzed the state of gene expression in OBX mice associated with apoptosis-programmed cell death, as well as ferroptosis, that are characteristic features of various forms of AD.

4. Apoptosis and ferroptosis.

The main manifestation of any neurodegenerative process is massive cell death. It has been established that AD is characterized by neuronal death, predominantly due to apoptosis pathway activation. Therefore, we analyzed the expression of apoptosis-related genes in the brains of OBX mice. The obtained data are presented in Fig. 5. The list of genes associated with apoptosis and ferroptosis processes was taken from the database http://deathbase.org/ and [44], respectively. The results indicate that the number of apoptosis-related genes that increased their expression was two times larger in comparison with genes with decreased expression. Obviously, in the brain of OBX mice, the hippocampus represents the structure where the largest number of genes with altered expression associated with apoptosis was noted.

The activation of the Dab2ip gene in all studied areas of the brain is noteworthy. This gene encodes a protein which acts as a scaffold protein implicated in the regulation of a large spectrum of both general and specialized signaling pathways. In particular, this protein modulates the balance between phosphatidylinositol 3-kinase (PI3K)-AKT-mediated cell survival and apoptosis-stimulated kinase (MAP3K5)-JNK signaling pathways [87].

Direct activation of apoptosis in OBX mice is associated with activation of the Casp9 gene in the hippocampus and the Apaf-1 gene in the cortex. Both genes are involved in the activation cascade of caspases responsible for apoptosis. It is also necessary to note the activation of the Hrk gene (DP5) in the cortex of OBX mice. This gene plays an important role in neuronal cell death [88]. On the other hand, the activation of the Bcl2l1 gene observed in the hippocampus of OBX mice may indicate a protective, compensatory effect against apoptosis, since this gene is a potent inhibitor of cell death [89].

Ferroptosis is a nonapoptotic form of cell death dependent upon intracellular iron [90]. Iron homeostasis disturbance has also been implicated in Alzheimer’s disease (AD), and excess iron exacerbates oxidative damage and cognitive defects [91].

In the hippocampus of OBX mice, only four genes related to this type of cell death changed their expression. Thus, Trf gene exhibited upregulation while Pcbp1, Ftl1, and Gpx4 genes were down-regulated (Figure 5). All these genes are associated with the maintenance of iron homeostasis, and their insufficient expression may lead to cell death by the ferroptosis pathway.

It is of note that drastic down-regulation of the Pik3r3 gene is observed in the brain of OBX mice (Figure 5). This gene under normal conditions, promoted hepatic fatty acid oxidation via PIK3R3-induced expression of Pparα [92]. Drastic inhibition of this gene in two brain areas of OBX mice should disrupt lipid metabolism which may lead to the induction of the ferroptosis process.

5. Changes in the transcription of genes characteristic for different cell types in the brain of OBX mice

Next, we analyzed the expression of genes in the cortex, hippocampus, and cerebellum related to the state and activity of different major types of cells in these brain regions and presented this in the form of histograms, where we plotted the number of genes that increased or decreased expression in OBX mice compared to SO animals in each of 6 major cell types: neurons, astrocytes, oligodendrocytes, microglia, endothelial cells and oligodendrocyte progenitor cells (OPC) (Figure 6). A complete list of genes expressed in different cell types in the compared brain structures is provided in Table S1.

The hippocampus again turned out to be the brain structure of OBX mice with the largest number of genes with altered expression related to the functioning of different types of cells of the nervous tissue. In the hippocampus and cortex, the largest proportion of genes, predominantly with increased expression, has been revealed for astrocytes, neurons, and progenitor cells, and in the cerebellum for microglia. An analysis of the synchronicity of changes of the same genes for different types of cells presented in Figure 6B demonstrated an interesting pattern: the same types of brain cells in the OBX mice were characterized predominantly by altered expression of different genes depending on the brain area studied.

When analyzing neuronal genes, we demonstrated that Map2, Camk4, and Lamp5 genes drastically reduced their expression in OBX mice, which according to the literature, is characteristic for AD [94-97]. The Arpp21 gene, which greatly decreases its expression in hippocampal neurons, is directly associated with a reduced dopamine receptor activation [98]. On the other hand, several genes with increased expression in OBX animals may also be associated with the development of AD-like pathology. Thus, up-regulation of both the Scn2b and Efr3a genes is associated with the impairing of learning and memory [99] and reduced survival of newly born neurons in the hippocampus by inhibiting the maturation [100]. The protein encoded by the Ap2a2 gene often co-localizes with neurofibrillary tangles and promotes their deposition [101].

Analysis of microglial genes in hippocampus of OBX mice with changed expression logFC < -0.8 enabled to conclude that these genes are involved in protein synthesis regulation (Eif5 - ribosomal initiation complex and chain elongations), metabolic coordination between cytoplasm and mitochondria (Ptp4a1), as well as in regulation of ionic homeostasis (Slc39a12).

When studying astrocytes, we observed down-regulation of expression of gene Naaa [102] and the Grm3 - gene of glutamate receptor metabotropic 3, associated with the accumulation of glutamate in the synaptic cleft leading to the development of neurotoxicity [103]. On the other hand, in the astrocytes of OBX mice, we revealed a significant increase in the expression of the Slc6a11 - gene encoding GABA transporter in the brain [104]. The activity of this gene which may ameliorate the hyperactivation of the glutamatergic system probably represents a compensatory reaction.

The observed increased expression of the GFAP gene (Glial fibrillary acidic protein) in OBX mice represents the manifestation of astrogliosis, a characteristic feature of AD [105, 106] as well as up-regulation of the Sox9 gene playing an important role in Aβ deposition being an important feature of the hippocampus in AD patients [52].

In microglia of OBX mice preferential up-regulation of large majority of genes was revealed. Characteristically, an increase in the expression of several highly specific genes for non-activated mouse microglia, such as Sall1 (transcription factor) was noted. The observed increased Trim8 expression in OBX mice, probably also represents the mechanism to protect microglial cells from cytotoxicity and inflammation [107].

Overexpression of the Ski gene demonstrated in all analyzed brain regions of the OBX mice, leading to the loss of neurons bearing NMDA receptors represents a manifestation of excitotoxicity apparently associated with excessive release of glutamate [108]. On the other hand, in microglia we revealed up-regulation of several genes activated by bulbectomy such as Nrp1 which activates the Syk gene to inhibit Aβ deposition and regulate microglial phagocytosis and disease-associated microglia (DAM) acquisition [55, 109].

All the above data enable to conclude that it is unlikely that an active process of neuroinflammation takes place in OBX animals. It is of note, that microglia cells are characterized by a drastic drop in the expression of genes involved in ribosome function (e.g. Rps27, Rpl22, Rps27a), transcription activation (Junb), protein transport (Rab11a) and neurogenesis (Hbp1, [110]).

The observed significant activation of genes in endothelial cells in OBX mice is also of interest because it may reflect changes in the permeability of the BBB. Besides, these cells as well as microglia cells exhibited reduced expression of ribosomal proteins (Rps27, Rps27a), splicing (Cwc15), protein transport and cell differentiation and degradation (Gabarapl2) , [111]. It is of note, that in the cortex and cerebellum of OBX mice, the expression of only 6 genes associated with oligodendrocytes changed their expression, while in the hippocampus, there were 16 of such genes. Dysregulation of the activity of these genes can lead to a decrease in the intensity of axonal myelination. Indeed, impaired axonal myelination in the brain at the early stages of AD development has been demonstrated [112, 113].

Therefore, we have shown that OBX mice are characterized by pronounced changes in the expression of genes associated with different types of nerve cells, and the direction of these changes often coincides with the patterns observed in the brains of AD patients.

6. Expression of genes involved in major depression in the brain of OBX mice

It should be noted that OBX mice and rats are often considered in the literature as a valid model of depression [27, 114, 115]. Figure 7 depicts differentially-expressed genes in the three brain structures of OBX mice that are associated with MD phenotype in humans (gene sets from [43]).

When analyzing differentially expressed genes in OBX mice, it is obvious that several of them coincided with genes responsible for the development of depression (including the genes Drd2, Map2, and the so-called canonical clock genes: Per2, Arntl, Nr1d1). These genes play an important role in the regulation of circadian rhythm. It is known that changes in their expression are accompanied by sleep disturbance, a characteristic symptom of both MD and AD [116, 117].

It is known that depression is the result of dysregulation in neurotransmitter functioning in different brain regions [118]. In OBX animals, as mentioned above, we observed pronounced changes in the expression of genes associated with the activity of dopaminergic (Th, Drd1, Drd2), serotonergic (Htr1a, Htr2c, Htr6), adrenergic (Adra2a) and cholinergic (Chrm1, Chrm4) brain systems. Probably the observed increase in the expression of the Sox9 gene encoding a transcription factor [119] represents a direct consequence of such disturbances. It is of note that the revealed changes in gene expression in OBX mice associated with the state of the peptidergic system, endorphin, and opiate systems are similar to those observed in MD, as well as observed hormonal dysfunction.

At the same time, while in OBX mice the synthesis of neurotrophic factors decreases, the level of intracellular calcium increases. Furthermore, in OBX animals the phosphorylation of tau protein is activated while apoptosis is induced on the background of astrogliosis. All these changes observed in OBX mice are also characteristic of animals with MD. Importantly, several genes depicted in Figure 7 are known to be involved both in MD and AD pathologies.

7. Determination of the subtype of AD-like pathology exhibited by OBX mice

Attempts are being made all the time to organize data into certain types or categories, both for pathologies like AD in humans and for the numerous animal models used to study this disease based on RNA-seq data [2]. We also analyzed DEG expression in OBX mice and found 20 genes (Table S2) belonging to the 101 “key regulators” identified in the article describing AD subtypes, that were obtained as a result of transcriptomic analysis of the hippocampus of AD patients from two large populations [2].

The performed comparison enables to conclude that the pattern of expression of OBX mice genome may be placed with caution into B2 subtype of AD (Table S2). Thus, protein degradation–related genes, involved in ubiquitination and polyubiquitination (Fbxo41, Fbx16), protein catabolism, the proteasome biogenesis, and proteins targeting for destruction, as well as genes responsible for the formation of vesicles and endosomes (Csrnp3, Cltc), were predominantly up-regulated in the hippocampus of OBX mice. The enhanced transcription in OBX animals was also shown for the genes responsible for intracellular and transmembrane traffic (Rab9b), influencing cell differentiation and phagocytosis (Syk). Thus, in an attempt to assign hallmarks of AD pathology in OBX mice to subtypes of human AD [2], we found a modest, but statistically significant correlation between RNA expression patterns in the hippocampus of OBX mice and B2 subtype of AD (R= 0.209, p=1.897906e-85) (Figure S3).

Next, we analyzed how the protein products of differentially expressed genes in different regions of the brain of OBX mice interact with several proteins of key AD-associated genes. The latter genes involved in the formation and processing of beta-amyloid, the processes of synaptic transmission and protein folding. The results of this analysis are depicted in Figure 8.

It can be seen in Figure 8 that the products of genes differentially expressed in different parts of the brain of OBX mice interact at the protein level with certain categories of genes involved in AD. This suggests that changes in the expression of certain genes in the brain of OBX animals lead to disruption of several key protein-protein interactions, which in turn triggers a cascade of changes leading to disruption of major regulatory systems in various parts of the brain. Interestingly, these systems include synaptic transmission, protein folding, beta-amyloid formation, etc.

The largest number of DEGs that changed their expression after OBX was observed in the hippocampus, which is consistent with the results of other studies that found a high correlation between the number of DEGs in the hippocampus and the progression of AD and including the intensity of amyloid plaque deposition [120].

From the results obtained, it follows that OBX mice are a model of a fairly common type of sporadic AD with manifestations of agitated depression observed in more than 30% of AD patients.

Intensive studies of AD in clinics and experiments exploring numerous models of this disease have made it possible to put forward several hypotheses for the genesis of this pathology. The generally accepted hypotheses are the amyloid cascade, the neurotransmitter hypothesis, the mitochondrial dysfunction hypothesis, neuroinflammation, and excitotoxicity hypothesis. Each of them is confirmed by abundant behavioral and biochemical data, as well as a description of a specific set of genes involved in the development of the pathological process in AD. Since the main goal of this study was to determine whether OBX animals represent a model of AD or MD, we analyzed gene expression in three brain regions of OBX mice and compared them with known sets of genes implicated in AD or MD. The removal of the olfactory bulbs has a strong effect on gene transcription in the brain. The hippocampus was the leader in this respect. These data agree with the abundant data accumulated in the process of AD investigation that demonstrated a high correlation between the number of DEGs in the hippocampus with the progression of AD and the intensity of amyloid plaque deposition [120].

The olfactory bulbectomy resulted in the activation of several signaling pathways leading to an AD-like neurological phenotype. Thus, the major affected biological processes according to GO enrichment are responsible for neurotransmitter secretion, cognition, learning, memory, neuropeptide signaling pathway, metabolic processes, ribosome processing and biogenesis, cell-matrix adhesion, etc. Characteristically, genes responsible for neuronal plasticity, neuropeptide signaling pathway, neurotransmitter transport, and secretion, as well as genes responsible for the regulation of glucose metabolism and WNT and Notch signaling were significantly up-regulated in the hippocampus of OBX mice. On the other hand, down-regulated genes were involved in protein synthesis and ribosome biogenesis.

The transcriptomic analysis of genes in the brain of OBX mice demonstrated that 36 out of 45 Alzheimer’s disease-associated genes changed their expression, while increased expression was demonstrated by genes directly associated with the development of a neurodegenerative process similar to AD (Fig. 3). In our analysis we have demonstrated unidirectional changes change in the expression of genes involved in deposition of beta-amyloid (e.g. up-regulation of the Sox9 gene) [52] or hyperphosphorylation and deposition of tau protein [121, 122], i.e. most of them exhibited increased expression. Interestingly, in the OBX brain, even several genes with reduced activity apparently contributed to the development of the pathology. Thus, a decrease in the expression of the prefoldin genes (Pfdn5 and Pfdn2), that perform the function of chaperones, was observed, probably contributing to the formation of beta-amyloid oligomers. It is interesting to note, that the enhanced expression of several genes in the brain of OBX mice was probably compensatory in nature leading to APP stabilization (e.g. Apba1, Apba2) or reducing Aβ load (Syk) by up-regulation of microglial phagocytosis (Piezo1) [55]. The presence of compensatory mechanisms that slow down the development of pathology is also characteristic for AD [123] and the very fact of activation of specific compensatory mechanisms can serve as an early diagnostic sign of the development of hidden pathology.

It is known that beta amyloid and tau protein deposits exhibit neurotoxic effects leading to neuron loss. It is also known that in AD the death of neurons occurs mainly through apoptosis [124]. In our case, the removal of the olfactory bulbs induces predominantly in hippocampus the synthesis of proapoptotic factors e.g. the expression of the Apaf-1 and Casp9 genes, that play a central role in the initiation of apoptosis [125, 126]. It is known that oligomeric Apaf-1 mediates the cytochrome c-dependent autocatalytic activation of pro-caspase-9 (Apaf-3), leading to the activation of caspase-3 and apoptosis. The increased level of the protein product of the Hrk gene, activated in OBX mice (DP5 [88]), also leads to activation of caspase-3 and, consequently, to neuronal cell death. In recent years, in addition to apoptosis, significant attention has been paid to other types of cell death, in particular, ferroptosis [44], which has been also implicated in Alzheimer's disease. Although only 4 genes associated with maintaining iron homeostasis changed their expression in OBX mice, downregulation of three of them is sufficient to induce ferroptosis (Pcbp1, Ftl1 и GPx4) [127, 128]. It is important to note that the inhibition of ferroptosis ameliorates brain damage in Alzheimer, Parkinson, and Huntington's diseases [129].

It is known that neurons loss observed in AD patients may be due to glutamate neurotoxicity. Therefore, we analyzed genes involved in the functioning of neurotransmitter systems that changed their expression after bulbectomy in the studied brain regions. The observed imbalance of glutamate/GABA systems with a deficiency of the inhibitory GABA system (i.e. reduced expression of the GABA A receptor-associated protein-like 2 genes) represents a characteristic feature of OBX mice transcriptome. Activation of the glutamatergic system in OBX mice was also evident based on the increased expression of metabotropic glutamate receptors and, apparently, a compensatory increase in the expression of the slc1a1 gene of the high-affinity glutamate transporters. Activation of this gene accompanies the accumulation of glutamate in the synaptic cleft and further development of glutamatergic neurotoxicity, leading to neuronal death [130].

Furthermore, a decrease in the expression of the mGlu3s receptor revealed in transcriptome of astrocytes in OBX mice may be associated with the accumulation of glutamate in the synaptic cleft and development of astrocyte-mediated excitotoxicity and neuronal death in OBX mice [103]. The overexpression of the Ski gene demonstrated in all brain areas in OBX mice is probably also associated with excessive release of glutamate, excitotoxicity and the death of neurons carrying NMDA receptors, which is typical for AD [108].

On the other hand, the increased expression of the Slc6a11 gene, encoding the GABA transporter, observed in astrocytes of OBX mice [104] may indirectly counteract the hyperactivation of the glutamatergic system. It is believed that an imbalance of these two systems may be a prerequisite for the development of AD [131]. Moreover, GABAergic dysfunction may contribute to the disruption of functional connections in the brain and thereby contribute to the development of AD [132]. The hypothesis of the genesis of AD associated with excitotoxicity caused by excess glutamate has become popular [133].

A decrease in the expression of genes, responsible for the synthesis of acetylcholine receptors M1 and M4, and the increased expression of the acetylcholinesterase enzyme gene represent another characteristic feature of OBX mice. It is necessary to emphasize, that a deficiency of the cholinergic system with hyperactivity of the acetylcholinesterase enzyme is a major symptom of AD and reflects the death of acetylcholine-synthesizing neurons in the nucleus basalis of the Meynert [134, 135]. A drop in the content of these receptors in human peripheral blood is used as a marker of AD [136]. Moreover, the development of pharmacological agents that enhance the expression of the Chrm1 and Chrm4 genes is one of the promising directions in AD treatment since the proteins they encode have neuroprotective activity and increase the survival of neurons in AD models [59, 60]. The observed increased expression of the acetylcholinesterase enzyme gene in the brain of OBX mice represents significant evidence of the validity of these animals as a model of AD.

At the present time the use of acetylcholinesterase enzyme blockers remains practically the only FDA approved method for improving memory in AD patients while the development of new agents with similar activity is a promising direction in pharmacology [63–66, 137]. It is believed that the deficiency of acetylcholinenergic system is associated with impaired cognitive functions in AD patients [61, 62].

Importantly, our transcriptomic data corroborate the results of our previous histoimmunochemical studies, that showed a decrease in the density of acetylcholine-synthesizing neurons in the forebrain basal ganglia of OBX mice [138].

The performed transcriptomic analysis also demonstrated the changes in the activity of the dopaminergic system in OBX mice. Thus, we revealed a decrease in the expression of dopamine type D2 receptors in the hippocampus, which is a characteristic symptom of both depression and AD [67, 139]. Previously, using a classical transgenic model of AD in 5XFAD mice, we also showed disturbances in the dopaminergic system, including the death of dopaminergic neurons, which is considered to be an early manifestation of AD [68]. The downregulation of genes encoding somatostatin (SSTs) in the brain of OBX mice can be also considered as the pathological signature of AD since low levels of both mRNA and correspondent protein are characteristic features of AD brain [69, 70].

Recently, the mitochondrial hypothesis of the pathogenesis of AD has taken the leading place in popularity [140, 141]. According to this hypothesis, energy deficiency caused by mitochondrial dysfunction is one of the major causes of AD development [142].

To this end, in the brain of OBX mice the vast majority of genes related to the functioning of mitochondria exhibited decreased expression, which indicates a disruption in the functioning of complexes 1 (the multisubunit NADH: ubiquinone oxidoreductase) and IV (cytochrome C oxidase) of the mitochondrial respiratory chain (down-regulation genes of the Nduf and Pet100 families), respectively), as well as complex V (ATP synthetase) (decreased expression of the Fmc1 gene). Deficiency of these mitochondrial complexes is a feature of several neurodegenerative disorders, including AD [143–145]. Data from transcriptomic analysis of the mitochondria state in OBX mice are consistent with our previously obtained results on impaired mitochondrial functions in these animals, similar to those occurring in transgenic familial mouse AD models and patients with sporadic AD [14]. Summarizing the discussion of the results of transcriptomic analysis of genes associated with the functioning of mitochondria and the rate of cell death in the brain of OBX mice it should be noted that although the number of genes associated with these processes and changing their activity in OBX mice was relatively small, the altered expression was observed in several major genes which directly indicates a deficiency of mitochondrial functions and the induction of apoptosis in the brain of OBX animals representing key symptoms of the development of Alzheimer's-like neurodegeneration. It is necessary to mention that recently we have demonstrated that intranasal administration of mitochondria isolated from control mice improves spatial memory in OBX mice [146].

Furthermore, we demonstrated that OBX animals are characterized by pronounced changes in the expression of genes associated with different types of nerve cells, and the direction of these changes often coincides with those observed in AD patients. Characteristically, the greatest changes for all six cell types were observed in the hippocampus. Thus, a decrease in the expression of neuronal genes (e.g. Map2, Camk4, Lamp5) directly indicated the development of Alzheimer’s-type neurodegeneration [97, 147]. Dysregulation of the Ap2a2 and Efr3a genes observed in OBX mice and characteristic of animal models of AD is often associated with neurofibrillary tangles [101] and inhibition of hippocampal neurogenesis characteristic for AD [100].

The performed analysis of gene expression in astrocytes and microglia, is of special interest because the transition of these cells into disease-associated state may induce neuroinflammation which plays the leading role in the genesis of AD [148]. However, our analysis of transcriptome of OBX mice brain regions failed to reveal clear-cut transcriptomic signatures of neuroinflammation with the only exception of activation of the pro-inflammatory cell surface marker galectin 3 gene (Lgals3 in cortex). An increased level of expression of the Trim8 gene in microglia [107], as well as activation of Syk signaling, which can interfere with the development of disease-associated microglia (DAM) suggest certain protection of microglial cells of OBX mice from cytotoxicity and inflammation.

On the other hand, the observed activation of Gfap gene in astrocytes of OBX mice is evidence of astrogliosis, characteristic for several neurodegenerative diseases, including AD [148]. It is of note, that increased expression of the Axl gene (tyrosine kinase receptor), in astrocytes in OBX mice is associated with inhibition of proinflammatory responses [149, 150]. The increased expression of the gene that controls the synthesis of glial fibrillary acidic protein (Gfap) in the brain of OBX mice indicates the validity of the model because this astrocytic cytoskeletal protein is a potential biomarker for AD [105, 106].

The demonstrated activation of genes associated with microglia may result in the release of glutamate, causing excitotoxicity and death of neurons bearing NMDA receptors, directly related to learning and memory [151]. The activation of the immune system of OBX mice takes place in the cerebral cortex judging by the high level of expression of genes involved in the interferon response (Ifitm3). It is well known that the induction of type 1 interferons, may be associated with neuron death and DNA damage [152]. Based on the accumulated data we conclude that neuroinflammation probably does not play an important role in the development of neurodegeneration in the brain of OBX mice. Characteristic feature of OBX mice is a dramatic decrease in the expression genes of ribosomal proteins in astrocyte, microglial cells and in particular genes for ribosomal mitochondrial proteins, which may lead to astrocytes and microglia dysfunction. It is well known that a drastic reduction of total protein synthesis is a characteristic feature of neurodegenerative process [153].

Analyzing the processes linked with OPC genes (oligodendrocyte progenitor cells), we discovered multidirectional changes associated both with the development of a neurodegenerative process and with a compensatory reaction in the brain of OBX mice. It is known, that genes of endothelial cells are able to maintain homeostasis and preserve the function of these cells even under conditions of bulbectomy and the development of neurodegeneration. We observed dysregulation the expression of genes in oligodendrocytes in the cortex and cerebellum of OBX mice, that may lead to a decrease in the intensity of axonal myelination, characteristic for the early stages of AD development [112, 113]. In this regard, it is interesting to note that the B2 subtype of AD in humans, which based on our data most closely corresponds to OBX mice, is also characterized by a marked down-regulation of genes in oligodendrocytes with up-regulation in other cell types, suggesting that a demyelinating process may take place in OBX mice.

Recently, special attention has been paid to comorbid diseases associated with AD, such as hypertension and diabetes, associated with disruption of the renin-angiotensin system (RAS) and insulin resistance. To this end, in OBX mice, we found a decrease in the expression level of the MAS1 receptor gene (Mas1). A shift in the balance of the two branches of the RAS, caused by a lack of MAS receptor (Masr) and angiotensin (1–7) against the background of an excess amount of angiotensin II, was often noted in sporadic AD, and its restoration has a positive effect in this pathology [154, 155].

Until now it was not clear whether major depression is a risk factor for AD, a symptom, a stress reaction in AD, or a comorbid disease. However, the very fact of comorbidity of AD, depression is a phenomenon long known. Serious studies have been devoted to the overlap between symptoms of depression and neurodegeneration processes in patients with mild AD [156]. The prevalence of symptoms of MD in Alzheimer's disease ranged widely from 22.5–54.4% [157]. However, due to the lack of convenient animal models, the question remained unclear. It was not clear whether depression arises as a psychological reaction to the disease and due to difficulties in the adaptation to AD, or depression and AD have common neurobiological basic mechanisms [158, 159]. It is of note, that transcriptional analysis of brain tissue in OBX mice revealed that some of the changes in receptor systems are more typical for MD, than for AD. Thus, the observed decrease in the expression of the adenosine receptor A2a gene (Adora2a) that we observed is a characteristic sign of depression [160, 161], while in AD patients, on the contrary, there is an increased expression of this receptor [74, 75, 162]. On the other hand, a decrease in the expression of dopamine type D2 receptors also demonstrated in the hippocampus in OBX mice is a characteristic feature of both depression and AD [67, 139].

Recently, using classical transgenic model of AD (APP/PSEN1-Tg mice) it was shown that depressive manifestations, such as a decrease in olfactory sensitivity represent an early stage of the pathology [163] Importantly, in the brain of OBX mice we observed the downregulation of the genes encoding somatostatin (SSTs) the dopaminergic) receptors, endorphin, and opiate systems, as well as genes associated with hormonal dysfunction. The analysis of transcription in the brains of OBX mice revealed up-regulation of genes associated with the regulation of circadian rhythms, cell migration, and impaired innate immunity, characteristic for the MD. Therefore, the transcriptomic analysis showed that in OBX mice, changes in the expression of genes responsible for the functioning of receptor systems are similar to those observed in AD, while changes characteristic of depression are less frequent and less pronounced. It is clear that OBX mice represent a model of complex neuropathology with elements of AD and MD. It is necessary in this context to underline that according to various studies [164], chronic use of antidepressants which has a protective effect in depression development in OBX mice is not able to reverse the development of neurodegenerative process in these animals including ventricular enlargement, hippocampal volume reduction, and neuronal loss, induced by bulbectomy.

In general, analysis of the functional significance of genes in the brain of OBX mice indicates that the balance of the GABA/glutamatergic systems is disturbed with hyperactivation of the latter, which leads to the development of excitotoxicity and induction of apoptosis on the background of severe mitochondrial dysfunction and astrogliosis. The synthesis of neurotrophic factors the OBX mice decreases, which leads to disruption of the cytoskeleton of neurons, an increase in the level of intracellular calcium, and activation of tau protein phosphorylation. The acetylcholinergic system is deficient on the background of hyperactivation of acetylcholinesterase. Importantly, the activity of the dopaminergic, endorphin, and opiate systems decreases, and hormonal dysfunction develops. Genes associated with the regulation of circadian rhythms, cell migration, and impaired innate immunity are activated in the model animals. All this occurs on the background of pronounced down-regulation of genes of ribosomal proteins. It is clear that OBX mice represent a model of complex neuropathology with elements of Alzheimer's disease and major depression. Apparently, OBX mice have neurobiological basic mechanisms responsible for complex symptomatology. Therefore, we demonstrated that OBX mice have a common transcriptomic signature associated with both AD and MD. The similarity of the genetic basis of these pathologies also determines the simultaneous manifestation of symptoms of AD and MD in OBX mice. Interestingly, dynamic changes in the gene regulation characteristic for depression were evident in mouse AD model before the onset of AD. From the presented data it follows that in OBX animals AD-like neurodegenerative process develops, including several elements of MD. Based on accumulated data this model can be tentatively attributed to subtype B2 of AD in humans.

Acknowledgements: The study was supported by the grant of the Russian Federation Government no 22-74-10050

Author Declarations:

Author Contribution: NVB, MBE designed the experiments; NVB, MBE, LNC, APR supervised the design and course of the experiments; NVB, VIK, DYZ, APR, CLN, AVC performed the experiments; NVB, MBE, LNC, APR performed the analyses and interpretation of the experiments; NVB, MBE, LNC, APR wrote the manuscript, NVB, MBE, LNC reviewed and edited the manuscript.

All of the authors read and approved the final manuscript.

Funding The study was supported by the grant of the Russian Federation Government no. 22-74-10050.

Competing Interest The authors declare no competing interests.

Data availability statement Data will be made available under the reasonable request

Ethics approval All animal experiments were conducted in agreement with the Provision and General Recommendation of Chinese Experimental Animals Administration Legislation and were approved by the Animal Ethics Committee in Institute (SLXD-20180912006, 12 September 2018) in compliance with Russian Federation legislation.

Consent to participate Informed consent was obtained from all individual participants and authors involved in the study

Consent to publish We confirming that consent to publish has been received from all participants involved in this stud

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A mouse model of sporadic Alzheimer’s disease with elements of major depression

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Abstract

Figures

Introduction

Materials and Methods

2.1. Animals

2.2. OBX Surgery

2.3. RNA Isolation, Library Preparation, and Transcriptome Sequencing

2.4. RNA data processing

Results

DISCUSSION

CONCLUSIONS

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1