1. Introduction

      Alzheimer’s disease (AD) is the most common dementia that progressively decline the memory and other mental functions in elderly individuals [1]. Currently, over 50 million people worldwide live with AD-related dementia, and this number is expected to increase to 152 million by 2050 (World Alzheimer Report 2020). AD is manifested by dementia in aged individuals [1]. AD-related dementia has huge economic consequences, with the total medical costs of dementia worldwide is estimated at $818 billion (World Alzheimer Report 2020). In addition to dementia, AD is associated with the loss of synapses, synaptic dysfunction, microRNA (miRNA) deregulation, mitochondrial structural and functional abnormalities, inflammatory responses, extracellular neuritic plaques, and intracellular neurofibrillary tangles (NFTs) [1–11]. Despite tremendous progress that has been made in better understanding the AD pathogenesis, there are still no detectable markers, drugs, and/or agents that can prevent AD or slow its progression. Search for a novel, non-invasive, early detectable biomarkers that are involved in AD and Alzheimer’s disease related disorders (ADRD) still ongoing. Several miRNAs have been reported that are involved in AD-related pathways and regulate AD pathogenesis via modulation of AD genes [6,12–18]. However, most of the studies are limited to in vitro cell culture treatment only [13–20]. Emerging studies revealed that MiRNA based interventions are ideal ones to develop therapeutics in AD. To understand the physiological functions of miRNA, it is important to generate both TG and KO mouse model, particularly for miRNAs associated with APP processing and amyloid beta (Aβ).

      MiR-455 is a member of a broadly conserved miRNA family expressed in most of phyla, including Mammalia and Primates. MiR-455 mostly implicated in human cancers and chondrogenic differentiations [21]. Recent reports unveiled the role of miR-455-3p in mitochondrial biogenesis through the upregulation of the PGC1α gene via regulation of novel HIF1an-AMPK-PGC1α, a signaling network [22]. For the first time, we investigated the connection of miR-455-3p in AD. Our lab was the first to investigate the relevance of miR-455-3p in AD [21,24–26]. Our global microarray analysis found higher expression of miR-455-3p in the serum samples from patients with AD relative to healthy controls [23]. Further, findings on AD postmortem brains, AD-Fibroblasts, AD B-lymphocytes (all from late onset AD; LOAD), AD cerebrospinal fluids, AD cell lines, and APP transgenic (Tg2576 strain) mice exhibited higher expression levels of miR-455-3p in AD cases compared to the samples from healthy controls [21,23–26].

      MiR-455-3p target several genes, most of them validated in cancers [21]. In relation to AD, APP was the most authenticated and one of the top predicted targets of miR-455-3p [25]. Overexpression of miR-455-3p construct reduces the expression of mutant APP cDNA in mouse neuroblastoma cells. The levels of full-length APP, C-terminal fragments of APP (C99 and C83) and the levels of Aβ1-40 and Aβ1-42 were also significantly decreased in cells by miR-455-3p. Further, overexpression of miR-455-3p reduces the toxic effects of Aβ on mitochondrial biogenesis, mitochondrial dynamics, synaptic activities, cell viability and apoptosis [25].

       Based on the in vitro findings, it is well established that high levels of miR-455-3p reduces Aβ pathology, enhances mitochondrial biogenesis, mitochondrial function, and synaptic activity, whereas our studies of reduced the endogenous miR-455-3p, showed increased Aβ, reduced mitochondrial biogenesis and reduced mitochondrial function. However, to confirm our in vitro findings, and understand the physiological functions of miR-455-3p, creation of overexpressed (TG) and depleted (KO) miR-455-3p mouse models is necessity. Mouse models are the best tools to study the molecular mechanism and impact of miRNAs in the disease process. Therefore, in the current study, using pronuclear injection and CRISPR technologies, for the first time, we generated both transgenic and knockout mouse models of miR-455-3p and characterized for lifespan extension, cognitive behavior, mitochondrial and synaptic activities.

2. Materials and methods

2.1 Animals

      The animal study was approved by Texas Tech University Health Sciences Center - Institutional animal care and use committee (TTUHSCIACUC). The wild type (WT) mice (C57BL6/J) aged 12 months were purchased from The Jackson Laboratory (Bar Harbor, ME). The microRNA-455-3p Transgenic (miR-455-3p TG; TG) and microRNA- 455-3p Knockout (miR-455-3p KO; KO) mouse models were generated in collaboration with Cyagen Biosciences, Santa Clara, CA, USA. The twelve months old, mice were divided into three groups-Group 1: WT mice, Group 2: TG and Group 3: KO heterozygous (KO+/-), we call these animals KO, from here on in the manuscript. All animals were housed under air-conditioned rooms at a constant temperature of 22 ◦C with a 12 h light/dark cycle and given access to water and food ad libitum.

2.2  Generation of miR-455-3p transgenic mouse model

      MiR-455-3p overexpressing TG mouse model was generated on a C57BL6 mice background, using a pronuclear plasmid injection of miR- 455-3p expression vector (pRP[Exp]-U6>hsa-miR-455-3p-CAG-EGFP) as shown in Supplementary Information Fig. 1 (SI Fig. 1) in collaboration with Cyagen Biosciences (Supplementary materials). Before, the generation of TG mice, the biological properties of miR-455-3p construct were tested in vitro using mouse neuroblastoma cells [25]. To generate TG model, we tagged miR-455-3p construct with the green fluorescence protein (GFP), in order to detect miR-455-3p transfection and the localization of GFP within cells [25]. The resulting TG mice founders did not show any kind of physical abnormality and lethality related to miR-455-3p transgene at any stage of life and TG animals viable and fertile. 

2.3  Generation of miR-455-3p knockout mouse model

      MiR-455-3p KO (±) mouse model for miR-455-3p suppression was generated using the CRISPR/Cas9 technology to knockout the endogenous miR-455-3p (Supplementary materials). Two pairs of gRNA constructs targeting the 3’ of mouse miR-455 and Cas9 mRNA were coinjected into fertilized mouse eggs to generate targeted knockout offspring (SI Fig. 2). F0 founder animals were identified by PCR, followed by sequence analysis, which were bred to wild type mice to test germline transmission and F1 animal generation. The deletion of 41 bp sequence of miR-455-3p was confirmed by sequencing analysis (SI Fig. 3). The resulting miR-455-3p KO positive founders were characterized by KO specific genotyping strategy (SI Fig. 2). The miR-455-3p TG and miR-455-3p KO confirmed pups were bred to obtain more pups (SI Fig. 4). The miR-455-3p KO pups did not show any kind of physical abnormality and lethality related to miR-455-3p deletion at any stage of life.

3.4  Impact of miR-455-3p on mRNA levels

      To determine mRNA levels of mitochondrial biogenesis, dynamics and synaptic genes, we performed qRT-PCR analysis of all mitochondrial  biogenesis, dynamics and synaptic genes in WT, TG and KO mice. The TG mice showed increased expression of biogenesis genes (PGC1α, Nrf1, Nrf2, TFAM), fusion proteins (Mfn1, Mfn2 and OPA1), synaptic and dendritic genes (SNAP25, PSD95 and MAP2) and reduced expression of fission genes (Drp1 and Fis1) in TG mice relative to WT and KO mice (SI Fig. 6). In KO mice, expressions of PGC1α, Nrf1, Nrf2, TFAM, Mfn1, Mfn2, OPA1, SNAP25, PSD95 and MAP2 were reduced compared to WT and TG mice, and Drp1 and Fis1 expression levels were increased relative to TG mice. These results further validate the positive roles of miR- 455-3p in regulation of mitochondrial biogenesis, dynamics and synaptic genes.

3.5  Impact of miR-455-3p on protein levels-immunoblotting analysis

      To determine the impact of miR-455-3p on mitochondrial biogenesis and dynamics activity, we analyzed the levels of mitochondrial biogenesis (PGC1α, Nrf1, Nrf2 and TFAM) and mitochondrial dynamics proteins (Drp1, Fis1, Mfn1, Mfn2 and Opa1) in WT, TG and KO mice brain tissues.

      Fig. 3A, shows the representative immunoblots with Fig. 3B showing the densitometry analysis. Our densitometry analysis shows the significantly increased levels of all four biogenesis proteins in TG mice relative to WT and KO mice (Fig. 3B). In KO mice, mitochondrial biogenesis protein levels were decreased significantly compared to WT mice.

      Next, we determined the protein levels of mitochondrial dynamics (fission proteins-Drp1 and Fis1) and (fusion proteins-Mfn1, Mfn2 and Opa1) in WT, TG and KO mice (Fig. 4A). The densitometry analysis shows that mitochondrial fissions protein Fis1 was reduced significantly in TG mice relative to WT (P = 0.043) and KO (P = 0.012) mice, but Drp1 did not show any significant change (Fig. 4B). The mitochondrial fusion protein Mfn1 showed significantly increased levels in TG mice relative to WT (P = 0.004) and KO mice (P = 0.0003). The Mfn2 protein levels did not show a significant change in TG mice though it was significantly reduced (P = 0.0063) in KO mice relative to WT mice. Further, Opa1 levels were also increased significantly in TG mice relative to KO mice (P = 0.005) (Fig. 4B).

      To understand the impact of miR-455-3p on synaptic and dendritic activities, we also assessed the levels of synaptic proteins- SNAP25, PSD95 and MAP2 in WT, TG and KO mice. Fig. 5A, shows representative immunoblots of SNAP25, PSD95 and MAP2 proteins in WT, TG and KO mice. The densitometry analysis reveals significantly increased levels of SNAP25, PSD95 and MAP2 proteins in TG mice compared to WT mice (Fig. 5B).

3.6  Impact of miR-455-3p on protein localization-immunostaining analysis

      To understand the localization of mitochondrial biogenesis proteins, we performed immunostaining analysis, focusing on hippocampal and cortical regions of WT, TG and KO mice (Fig. 3C). Fluorescence intensity quantification shows significantly increased fluorescence immunoreactivities of all four proteins PGC1α, Nrf1, Nrf2 and TFAM in TG mice relative to WT and KO mice (Fig. 3D).

      We also performed the immunostaining analysis of mitochondrial dynamics proteins in WT, TG and KO mice brains (Fig. 4C). Most of the proteins showed similar trends as seen by immunoblotting analysis. The fluorescence intensities of Drp1 fission protein were found to be significantly increased in KO mice relative to TG (P = 0.021) and WT mice (P = 0.002). The fluorescence intensity of all fusion proteins Mfn1, Mfn2 and OPA1 were found to be significantly increased in TG mice relative to WT and KO mice (Fig. 4D). These results signify that miR- 455-3p overexpression positively regulates the mitochondrial structural and functional activities.

      The immunostaining data of synaptic proteins also shows increased levels of SNAP25, PSD95 and MAP2 in TG mice relative to WT and KO mice (Fig. 5C and D). These results concur with our mRNA data.

3.7  Impact of miR-455-3p on brain cell types

      To determine the impact of miR-455-3p on brain cell specific populations and differentiation-neuron, astrocytes and microglia, brain sections were immunostained using cell type-specific markers - NeuN, GFAP and Iba1 respectively. Fig. 6A, shows the representative immunostaining images of neurons, astrocytes and microglia in WT, TG and KO mice. Fluorescence intensity quantification shows significantly increased immunoreactive intensity of NeuN in TG mice relative to WT (P = 0.002) and KO mice (P = 0.0003) (Fig. 6B). The KO mice showed significantly reduced levels of NeuN relative to WT mice (P = 0.013). The astrocyte population (GFAP expression) was significantly reduced in TG mice relative to both WT (P = 0.0004) and KO mice (P = 0.005). Whereas astrocyte and microglia populations were found to be increased significantly in KO mice relative to TG (P = 0.012) and WT mice (P = 0.027) (Fig. 6B). These observations confirm that miR-455-3p overexpression increases neuronal populations and knockdown of miR-455- 3p enhances astrocyte and microglial activity.

3.8   Impact of miR-455-3p on dendritic spine density

      To determine the impact of miR-455-3p on dendritic morphology i.e., length and spines number, we quantified dendritic length and number of spines using Golgi-Cox staining in the hippocampi of 12-monthold WT, TG and KO mice. We compared the data within three groups of animals. Fig. 7A shows the representative images of dendrites in WT, TG and KO mouse brains covering both cortex and hippocampus areas at 4×, 10×, 20× and 60× magnifications. Hippocampal neurons from TG mice showed significant visual differences with dense and elongated dendrites compared to both WT and KO mice. The measurement of dendritic length was significantly increased in TG mice relative to WT (P = 0.0005) and KO mice (P < 0.0001) (Fig. 7B).

      Further, we quantified the number of dendritic spines using high magnification (60×) images in WT, TG and KO mice as shown in Fig. 7A. The number of dendritic spines were found to be significantly increased in TG mice compared to WT (P = 0.0006) and KO mice (P < 0.0001) (Fig. 7C). Interestingly, we also found significant differences in the dendritic numbers and lengths in KO mice compared to WT mice. These observations indicate that miR-455-3p overexpression enhances both dendritic length and number of dendritic spines, which are reduced in knockout miR-455-3p mice. These results confirm the significant positive impact of miR-455-3p on hippocampal neurons with dendritic morphology and quality.

3.9  Impact of miR-455-3p on mitochondrial morphology

      To determine the impact of miR-455-3p on mitochondrial number and length, we used transmission electron microscopy on cortical and hippocampal tissues from 12-month-old WT, TG and KO mice. We analyzed the data in terms of mitochondrial number and length in all three groups of mice. Fig. 8A shows representative images of mitochondrial morphology in cortical and hippocampal areas of WT, TG and KO mice brains. Fig. 8B shows significantly reduced mitochondrial number in the cortex of TG mice relative to WT (P = 0.013) and KO mice (P = 0.0001) (Fig. 8B). Interestingly, KO mice also showed significantly increased mitochondrial (fragmentation) numbers (P = 0.002) relative to WT mice.

      Additionally, mitochondrial length was significantly increased in TG mice relative to WT mice (P = 0.038), however we did not see any significant differences in mitochondrial length in KO mice compared to TG mice (Fig. 8C).

     Next, we examined the mitochondrial morphology in WT, TG and KO mice hippocampus area (Fig. 8A). Fig. 8D shows significantly reduced mitochondrial number in TG mice relative to WT (P = 0.042) and KO mice (P = 0.0002). However, mitochondrial length was significantly increased in TG mice relative to WT (P = 0.004) and KO mice (P = 0.0001) (Fig. 8E).

     The KO mice showed poor mitochondrial quality, in terms of mitochondrial fragmentation compared to WT and TG mice. These observations confirm that high levels of miR-455-3p improve the mitochondrial morphology in terms of number and length in both the cortex and hippocampus.

3.10  Impact of miR-455-3p on synapse numbers

      We also examined the impact of miR-455-3p on synapse organization and numbers in both the cortex and hippocampus of WT, TG and KO mice. Fig. 8F shows the synapse locations and organization in WT, TG and KO mice cortex. As shown in Fig. 8G, the average synapse numbers were significantly increased in TG mice cortex relative to both WT (P = 0.0009) and KO mice (P = 0.0073). Fig. 8F shows the synapse locations and numbers in WT, TG and KO mice hippocampus. Quantification shows significantly increased synapse numbers in TG mice relative to KO (P = 0.028) but we did not find significant difference in TG vs WT mice (Fig. 8H). These observations indicate that miR-455-3p is involved in synapse assembly and junction formation.

4.  Discussion

      Mouse models are the frequently used research tools to study the molecular basis of disease and to develop therapeutic strategies. Several AD mouse models (TG, KO, Knock-in) have been used to understand disease process from birth to terminal stages and also to develop therapeutic drugs. AD mouse models have been used to test anti-AD agents and to study the impact of genetic mutations and gene products, proteins via genetic crossings with other TG and KO mouse models [29,30].

      MiRNA mouse models are emerging tools to study the protective and/or deleterious effects of miRNAs in human diseases [31,32]. Since, single miRNA, for example miR-455-3p is implicated in multiple human diseases such as cancers and chondrogenesis via modulating a bunch of potential target proteins [21]. Recently, our lab discovered the properties of miR-455-3p in AD [21]. Based on the critical roles of miR-455-3p in AD and anti-AD characteristics, it was warranted to develop miR-455-3p mouse models. Other than AD, generation of miR-455-3p mouse models could be useful for miR-455-3p dependent other human diseases also.

      MiRNAs molecules have been identified as strong modulators of human genes and/or proteins endogenously at genetic and epigenetic levels [33]. Several miRNAs have been studied previously in AD that showed promising results against AD genes/proteins via modulation of key AD related genes [13–20]. However, most of miRNA(s) studies did not go longer in-depth analysis due to lack of suitable mouse model(s) and possible interventions. Quite a few studies showed the miRNAs intervention using either direct oral/nasal miRNAs complex administration or by injections [34]. Again, these studies had several limitations, 1) dose and route of miRNA administration, 2) miRNA complex stability, 3) immune reactivity of miRNAs encapsulated particles, 4) half-life of exogenous miRNA complex inside the body and 5) degree of effectiveness. To overcome these challenges and technical barriers, the generation of miRNA mouse model is ideal and provides detailed molecular mechanisms and therapeutic potentials of a particular miRNA. Based on the previous miR-455-3p studies [23–26] and its promising anti-AD properties [21], the current study was attempted to develop miR-455-3p TG and KO mouse models.

      In the current study, miR-455-3p TG and KO models were success fully generated by using pronuclear injection of miR-455-3p transgene to mouse embryo and CRISPER/Cas9 knockout techniques. Both TG and KO mice were characterized by genotyping, sequencing, miR-455-3p qRT-PCR and immunoblotting studies. Both mouse models displayed normal phenotypic characteristics and we did not observe any serious deformities due to miR-455-3p transgenes insertion and depletion.

     Lifespan extension: We followed TG, KO and non-transgenic and non-KO WT mice from birth to terminal stages of life, and our survival data revealed that TG mice lived 5 months longer than age-matched non-transgenic WT mice; on the other hand, KO mice survived 4 months shorter than their non-transgenic WT counter parts. These observations indicate overexpression of miR-455-3p extend overall lifespan, in other words, miR-455-3p has protective properties of brain. As described, our current study focused on brain, it is worthwhile to study other organs, such, heart, lung, liver, kidney, and skeletal muscle, in order to understand protective properties, how miR-455-3p protects different peripheral organs. Based on our current study, endogenous full-length APP mRNA and protein levels were reduced in TG-miR-455-3p mice, and these were increased in KO mice, it is possible that reduction of fulllength APP is protective to the brain. The dramatic increase of endogenous APP levels in the brains of KO mice indicates that KO mice are important to study APP related toxicities in AD.

      If miR-455-3p is capable of reducing full-length APP and its toxicities in mice, it is important to cross miR-455-3p TG and KO with mouse models of AD, particularly mice with intact 3’ UTR, say newly developed humanized Ab-KI mice. The resulting double mutant mice TG miR-455- 3p X hAb-KI predicted to live longer & show reduced cognitive decline and APP and its c-terminal derivative toxicities of mitochondria and synaptic activities. On the other hand, double mutant KO miR-455-3p X hAb-KI mice expected to accelerate AD pathologies, increased cognitive decline and reduced lifespan.

      Cognitive behavior: The main physiological hallmark of AD pathogenesis is cognitive deficits, so we examined the miR-455-3p effect on mice cognitive behavioral activities. The TG mice showed better cognitive learning and memory compared to age matched WT mice in MWM test. We also observed that KO mice did not perform well on MWM test. These results showed that high endogenous miR-455-3p improves cognitive functions. To understand the molecular mechanism for cognition improvement by miR-455-3p, we studied the synaptic proteins, synapse assembly organization, synapse numbers and dendritic spine density of cortical and hippocampal neurons. Since healthy and active synapse are the key for a typical synaptic activity [35–38].

      Synaptic and dendritic activities: The TG mice demonstrated increased levels of SNAP25, PSD95 and MAP2 proteins and essentially increased synapse numbers in both cortex and hippocampus. In addition, the dendritic spine density of hippocampal neurons was also increased in miR-455-3p TG mice. However, it is not clear how miR-455- 3p upregulation enhances synaptic proteins and synaptic activity. It is well established that cognitive function was improved by increased synaptic proteins [37] and by upregulation of PGC1α [39]. Recent studies showed the improvement of cognitive function mediated by miR-455-3p upregulation via suppression of Histone deacetylase 2 (HDAC) and EphA4 [40,41]. High levels of miR-455-3p reduces the neurotoxicity by suppressing the EphA4 expression in hippocampal neurons [41]. Other study found the relevance of miR-455-3p in restoring neurological function and improving memory deficits in traumatic brain injury (TBI) [40]. Actually, cinnamic acid as a treatment of TBI increases the expression of miR-455-3p, which in-turn suppressed the HDAC2 and reduce memory impairments. Our findings are in the line with these published reports and support our current findings.

      Cell type analysis: We studied the impact of miR-455-3p on brain cells differentiation. Interestingly, neuronal populations were found to be increased in TG mice as shown by NeuN staining. Even increased levels of other neuronal marker MAP2, in TG mice again confirmed the increased neuronal population by miR-455-3p over expression. Interestingly, astrocyte population was significantly reduced in TG mice compared to both WT and KO. On the other side, knockdown of miR-455-3p showed reduced levels of neuronal staining and KO mice showed the increased microglia and astrocyte expression.

      Mitochondrial structure and function: We also examined the miR- 455-3p impact on mitochondrial structure and function. Since mitochondrial structural and functional abnormalities are primarily involved in disease progression and synaptic dysfunction in AD [42,43]. As a parameter of mitochondrial quality, the TEM analysis for the evaluation of mitochondrial number and length showed improved mitochondrial organization in miR-455-3p TG mice. The improved mitochondrial quality was correlated with the levels of mitochondrial biogenesis proteins in TG mice. All four biogenesis proteins (PGC1α, NRF1, NRF2, and TFAM) were elevated in TG mice and the levels of dynamics proteins (DRP1 and FIS1, OPA1, Mfn1, and Mfn2) were positively regulated by miR-455-3p. However, it is argued that how miR-455-3p regulate the mitochondrial functions. A well-known mechanism is modulation of HIF1a by miR-455-3p [22]. On the other side HIf1a interaction with PGC1α is well established in mitochondrial function [44,45]. Therefore, miR-455-3p mediated HIF1a and PGC1α are the key drivers that controls mitochondrial function in TG mice.

      In summary, based on the miR-455-3p properties, current study is focused on the generation and characterization of miR-455-3p TG and KO mouse models to understand the molecular mechanism of miR-455- 3p in mice survival, APP protein, brain cells proliferation, synapse assembly formation, mitochondrial function, synaptic activity and mice cognitive behaviors. MiR-455-3p TG mice showed the improved lifespan, neuronal growth, synapse, cognitive function and overall mitochondrial and synaptic functions. While miR-455-3p KO mice exhibited reduced lifespan, neuronal growth, memory, mitochondrial and synaptic function.

      In conclusion, miR-455-3p exhibits positive impacts on neuronal activity in several ways and improve overall brain functions. Further, genetic crossing study is needed to understand the protective effect of miR-455-3p on the mutant APP proteins using hAb-KI and APP TG mice lines. Additionally, these mouse models could be ideal tool understand the molecular mechanism of miR-455-3p in AD and other human

Declaration of competing interest

      We would like to inform you that we have a pending patent ‘MicroRNA-455-3p as a Potential Peripheral Biomarker for Alzheimer’s Disease (US 20200255900)’ related to the contents of our manuscript.

Acknowledgements

     The authors would like to thank NIH for funding various projects -R01AG042178, R01AG47812, R01NS105473, AG060767, AG069333, and AG066347 to (P.H.R) and K99AG065645 to (S.K.).

Appendix A. Supplementary data

     Supplementary data to this article can be found online at https://doi. org/10.1016/j.redox.2021.102182.

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This article is excerpted from the Redox Biology 48 (2021) 102182 by Wound World.