Arginase Inhibition Supports Survival and Differentiation of Neuronal Precursors in Adult Alzheimer’s Disease Mice

Adult neurogenesis is a complex physiological process, which plays a central role in maintaining cognitive functions, and consists of progenitor cell proliferation, newborn cell migration, and cell maturation. Adult neurogenesis is susceptible to alterations under various physiological and pathological conditions. A substantial decay of neurogenesis has been documented in Alzheimer’s disease (AD) patients and animal AD models; however, several treatment strategies can halt any further decline and even induce neurogenesis. Our previous results indicated a potential effect of arginase inhibition, with norvaline, on various aspects of neurogenesis in triple-transgenic mice. To better evaluate this effect, we chronically administer an arginase inhibitor, norvaline, to triple-transgenic and wild-type mice, and apply an advanced immunohistochemistry approach with several biomarkers and bright-field microscopy. Remarkably, we evidence a significant reduction in the density of neuronal progenitors, which demonstrate a different phenotype in the hippocampi of triple-transgenic mice as compared to wild-type animals. However, norvaline shows no significant effect upon the progenitor cell number and constitution. We demonstrate that norvaline treatment leads to an escalation of the polysialylated neuronal cell adhesion molecule immunopositivity, which suggests an improvement in the newborn neuron survival rate. Additionally, we identify a significant increase in the hippocampal microtubule-associated protein 2 stain intensity. We also explore the molecular mechanisms underlying the effects of norvaline on adult mice neurogenesis and provide insights into their machinery.


Introduction
The adult murine brain continuously generates neuronal progenitor cells (NPCs) in the subventricular zone (SVZ) and subgranular zone (SGZ) of the hippocampal dentate gyrus [1]. differentiation and survival. We also explore the molecular mechanisms underlying the effects of norvaline on adult neurogenesis and provide insights into their machinery.

Strains of mice and treatment
Homozygous 3×Tg mice, harboring PS1(M146V), APP(Swe), and tau(P301L) transgenes, were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and bred in our animal facility. These mice exhibit memory deficits associated with amyloid plaques deposition and tangle pathology [40].
Randomly chosen, male 4-month-old transgenic mice and age-matched male C57Bl/6 mice (wildtype) were divided into four groups (

Tissue preparation and slicing
Four animals from each group were deeply anesthetized, with an intraperitoneal injection of 0.2 ml Pental (CTS Chemical Industries, Kiryat Malachi, Israel). The animals were perfused transcardially with ice-cold phosphate buffer saline (PBS), followed by ice-cold paraformaldehyde 4% in PBS. The mice were decapitated, and their brains were carefully removed and fixed in 4% paraformaldehyde for 24 hours and then were transferred to 70% ethanol at 4°C for 48 hours. The tissues were dehydrated and paraffin-embedded. The paraffin-embedded tissue blocks were chilled on ice and sliced on Leica RM2235 manual rotary microtome to a thickness of four µm. Then, the sections were mounted onto gelatin-coated slides, dried overnight at room temperature, and stored at 4 °C in slide storage boxes.

Quantitative immunohistochemistry
We studied neurogenesis within the dentate gyrus of the adult mice hippocampal formation by means of immunohistochemistry. Staining was accomplished on the Leica Bond Max system (Leica Biosystems Newcastle Ltd, UK). Brain sections were dewaxed and pretreated with the epitope-retrieval solution (ER, Leica Biosystems Newcastle Ltd, UK), and then incubated for 30 minutes with primary antibodies. A Leica Refine-HRP kit (Leica Biosystems Newcastle Ltd., Newcastle upon Tyne, UK) served for hematoxylin counterstaining. The omission of the primary antibodies served as a negative control.
Quantitative immunohistochemistry was accomplished using plane-matched coronal brain sections stained with appropriate antibodies, which produced a brown-colored end-product visible under a bright-field microscope. The coronal brain sections cut at 25 µm intervals throughout the brain per mouse (1.8-1.9 mm posterior to bregma) were used for the analysis.

Doublecortin labeling and staining
Newly formed neurons were first labeled with doublecortin (DCX), whose expression is specific for newly generated neuronal cells [42]. We utilized the polyclonal antibody GTX134052 (GeneTex, Irvine, California, USA) diluted at 1:500, to detect doublecortin protein, and quantified the number of DCX-positive neurons and the level of their stain intensity with Zen 2.5 software.
The density of neural progenitors (DCX+ cells) in the dentate gyrus was calculated in a circle with a diameter of 385 µm 2 and presented as the number of DCX+ cells per square mm. DCX+ objects with the surface area greater than 10 µm 2 were taken into account.

Polysialylated neuronal cell adhesion molecule staining
Polysialic acid (PSA) is a homopolymer whose primary carrier in vertebrates is NCAM [43].
Commonly, DCX hippocampal expression is temporally in-frame with PSA-NCAM expression [42]. The molecule is exceedingly expressed in the brain during development; still, in the adult murine brain, newborn granule cells of the dentate gyrus highly express PSA-NCAM as well [44].
Accordingly, PSA-NCAM is a popular marker to study structural plasticity and neurogenesis in mammals.
In order to detect PSA-NCAM, we utilized the monoclonal antibody 12E3 #14-9118-82 (eBioscience™, Thermo Fisher Scientific, Waltham, Massachusetts, USA) diluted at 1:100. We quantified the PSA-NCAM-positive surface area and intensity within the dentate gyrus. Zen 2.5 with a preset threshold was used to measure these parameters in a circle with a diameter of 220 µm 2 .

Microtubule-associated protein 2 staining
Microtubule-associated protein 2 (MAP2) is the most abundant brain MAP, which is predominantly expressed in dendrites and neuronal cell bodies during neurite outgrowth and dendritic branching [45].

Imaging and quantification
The brain sections have been viewed under an automated upright slide scanning microscope Axio Scan.Z1 (Zeiss, Oberkochen, Germany). The images were captured with 20×/0.8 and 40×/0.95 objectives at z-planes of 0.5 µm. An Axio Imager 2 Upright ApoTome Microscope was used to capture images with 100×/1.4 oil immersion objective.
Image analysis was carried out using ZEN Blue 2.5 (Zeiss). A fixed background intensity threshold was set for all sections representing a single type of staining. In order to create high resolution data, the image deconvolution technique of entire z-series, with ZEN 2.5, was utilized. A computer-driven analysis was performed at each of the counting frame locations.
The surface of the immunoreactive area, above the preset threshold, was subjected to the analysis.
The image densitometry method was applied to quantify the amount of staining in the specimens.
The mean stain intensity of the specific channel was measured and presented as the average value for each treatment group.

Tissue sampling, RNA extraction, reverse transcription, and real-time polymerase chain reaction
Five animals per group were rapidly decapitated with scissors. Their brains were carefully removed, and entire hippocampi were sampled. Total RNA was isolated from the left hippocampi 4444602 Rev. C) in a 10 μL volume using a five ng cDNA template. PCR was run, and the data was analyzed in the StepOnePlus system installed with StepOne Software v2.3 (Applied Biosystems, Foster City, California, US). The quantification was performed using the comparative Ct (ΔΔCt) method [46].

Statistical analysis
Statistical analysis was conducted with GraphPad Prism 8.0 for Windows (GraphPad Software, San Diego, CA, USA). The significance was set at 95% of confidence. The two-way ANOVA test was used to demonstrate whether the genotype, the treatment, or the interaction between both factors have an impact upon the phenotype. The two-tailed Student's t-test was performed to compare the means of two groups. The Kolmogorov-Smirnov test served to evaluate the normality of the data distribution. All data are presented as mean values. Throughout the text and in plots, the variability is indicated by the standard error of the mean (SEM).

3×Tg mice show a significantly reduced DCX-positive NPC density, compared to WT, which demonstrate a dissimilar phenotype, and is unaffected by arginase inhibition
Typically, in mice, the newborn cells of the SGZ migrate into the granule layer and extend their dendrites into the molecular layer [42]. Immunohistochemical staining for DCX of coronal serial sections through the dentate gyrus efficiently revealed newly generated cells.
DCX is a microtubule-associated phosphoprotein, which efficiently labels late mitotic neuronal precursors and early postmitotic cells [47], and is widely used as a reliable marker for newly-born neurons in the adult hippocampus [48]. DCX-positive cells express other early neuronal antigens but are deficient of antigens specific for glia or apoptotic cells [49]. DCX expression is profound in dendrites; accordingly, newly-born neurons' absolute number and dendritic growth can be efficiently evaluated with DCX immunostaining technique.
Seven-month-old male WT animals show characteristic patterns of DCX expression (Fig 1a). The observed DCX-positive cells are distributed heterogeneously within different regions of the dentate gyrus and are arranged in clusters (Fig. 1a, b). Remarkably, in the WT, the vast majority of the hippocampal DCX-positive neurons are situated in the SGZ; still, a substantial part of them are visible in the granular layer (Fig. 1a, b insets). These bipolar cells demonstrate extensive dendritic growth into the molecular layer.
In contrast, the 3×Tg mice DCX-positive cells do not exhibit extensive dendrites, and are marginally present in the granular layer (Fig. 1c, d). Two-way ANOVA test reveals a significant effect of genotype on DCX positivity with a significant (p<0.0001; F1, 28 = 203.2) reduction in the levels of DCX positive surface area (Fig. 1f), cell density (Fig. 1e), and mean stain intensity (Fig.   1g) in 3×Tg mice as compared to WT age-matched animals. The treatment factor had no significant influence upon these parameters. Additionally, the interaction accounted for less than 0.1% of the total variance.

Norvaline caused an escalation of the PSA-NCAM levels in the hippocampi of 3×Tg mice, as evidenced by an increase in immunopositive surface area and stain intensity
In order to corroborate the norvaline effects upon the rate of newly generated neurons survival and differentiation rate in adult 3×Tg mice, we tested the hippocampal levels of PSA-NCAM expression via immunohistochemistry. We observed a significant effect of the treatment on PSA-NCAM expression in SGZ, which is characterized by an increase in the levels of stain intensity (Fig. 2d) and the immunopositive surface area (from 0.76±0.2% to 1.86±0.22%) (Fig. 2c). Of note, PSA-NCAM-positive cells are scarcely present in the SGZ of 3×Tg mice and do not penetrate the granular layer (Fig. 2a). In contrast, these neurons are frequent in the SGZ and the granular layer of the 3×Tg mice treated with norvaline (Fig. 2b).

Norvaline rescues neuronal and dendritic loss in 3×Tg mice, as evidenced by MAP2 staining
The dynamic behavior of microtubules is crucial during cell division. MAP2 is a neuron-specific protein stabilizing dendritic microtubules; thus, it serves as a reliable neuronal marker [50].
MAP2-positive neurons possess relatively large cell bodies (more than 20 µm in diameter) and one or more dendrites (50 µm or longer) [51].
We measured the mean stain intensity of the hippocampal MAP2-positive objects and the immunopositive surface area. MAP2-positive objects were quantified in the cornu ammonis I (CAI) (Fig. 3e,f) and hilus areas (Fig.3 c,d). Norvaline-treated brains demonstrated robust MAP2 signal, while vehicle-treated brains exhibited a decrement in MAP2 signal as evidenced by twotailed Student's t-test. We observed a significant effect of the treatment (p=0.0002, t=4.403, df=22) on MAP2-positive area (with more than three-fold increase) in the CA1 region (Fig. 3h).
Stain intensity also demonstrated a significant elevation in CA1 (Fig. 3i). Analysis of the same parameters in the hilus area did not reveal any significant effect, though stain intensity increased with a p-value of 0.059 (Fig. 3g).

Norvaline escalates the transcription levels of CCL11
Eosinophil chemotactic protein (CCL11) has been shown to promote the migration and proliferation of NPCs in vivo and in vitro [52]. In order to decipher the mechanisms of observed treatment-associated differences in adult neurogenesis, we examined the transcription levels of this β-chemokine in the hippocampi of 3×Tg mice.
Remarkably, the levels of CCL11 mRNA are 79% higher in the norvaline treated mice than in controls. The Student's t-test demonstrated the significance of the difference between the means of control and treated animals (p=0.0415, t=2.425, df=8) (Fig. 4).

Discussion
Adult neurogenesis is a complex physiological process that plays a crucial role in the maintenance of normal cognitive functions. This process consists of progenitor cell proliferation, newborn cell migration, and eventually, their maturation [42]. Newborn neurons incorporate into existing functional networks. They are identifiable via various labeling techniques. Dentate gyrus progenitor cells proliferate in the SGZ and migrate into the granular layer of the dentate gyrus.
Then, they differentiate and become postmitotic cells with a different phenotype. The new neurons extend their axons to the hippocampal CA3 region and send dendrites to the molecular layer, which functionally integrates them into the hippocampal network [42].
It is worth highlighting that the resident hippocampal precursors are progenitor cells, which are capable of proliferation and multipotential differentiation, though, are incapable of self-renewal Control Norvaline 0 1 2 3 mRNA relative levels (fold change) [53]. Thus, the extra-hippocampal stem cells generate progenitor cells, which then migrate to the dedicated neurogenic area (SGZ) and proliferate there to produce progeny that differentiate into a population of DCX-expressing newborn neurons. The immature neurons, which express both DCX and PSA-NCAM, decorate the thin lamina underlying the SGZ, and migrate into the granule cell layer [42] (Fig. 1, 2). These cells undergo synaptic integration by sending extensive processes towards the molecular layer and CA3 area, and eventually become typical postmitotic cells.
Adult neurogenesis is an extremely vulnerable process, which is prone to alterations under numerous physiological and pathological conditions. Several lines of evidence suggest a substantial impairment of neurogenesis in AD, which is one of the earliest pathological characteristics of the disease, and its manipulation has been pursued as a potential therapeutic strategy [54].
Various AD animal models show age-dependent neurogenesis deficiency. Decreased proliferation of the hippocampal progenitor cells has been demonstrated in APPswe/PS1dE9 transgenic mice [55]. 3×Tg mice are also characterized by meaningfully impaired adult neurogenesis [3].
Remarkably, various approaches are capable of inducing neurogenesis in adult rodents, including environmental enrichment and enhanced physical activity [56]. Furthermore, numerous studies report a reversal of the decline in neurogenesis in transgenic AD murine models, including 3×Tg mice, as a corollary of different treatment strategies [57][58][59].
Growing empirical evidence indicates a unique role of NO in adult neurogenesis [60].
Accordingly, several agents have been successfully trialed with a rationale to increase brain NO levels, including arginine [61] and NO-donor supplementation [6]. In this study, we investigated the effects of a different NO-inducing approach upon adult neurogenesis. We utilized an arginase inhibitor, the non-proteinogenic amino acid norvaline, to promote adult neurogenesis in a murine model of AD. We assessed the neurogenesis rate by quantitatively evaluating the proliferation and differentiation of NPCs in the dentate gyrus SGZ. We applied several popular neuronal markers to characterize the different stages of neurogenesis by means of immunohistochemistry.
Previously, we used an advanced proteomics assay to evidence a significant (by 43%) elevation in NCAM protein levels following norvaline treatment in 3×Tg mice brains [15]. In the present study, we scrutinized the spatial patterns of PSA-NCAM hippocampal expression in relation to the treatment. Of note, PSA-NCAM-positive immature neurons have been shown to contribute to the early steps in adult hippocampal neurogenesis, such as proliferation and differentiation [62].
Consequently, PSA-NCAM is used as both a survival and a migration-associated neuronal marker [63]. This biomolecule is required for newly generated neuron survival in vitro [64] and in vivo [65]. Likewise, the role of PSA-NCAM in migration regulation and in the stimulation of newly generated neuron processes outgrowth has also been suggested [66]. Our methodology revealed a significant increase in the hippocampal granular layer PSA-NCAM positive surface area (Fig. 2 c) and intensity (Fig. 2 d) following the treatment, which implies improvement in the newborn neuron survival rate and accords with our previous results.
It is worth emphasizing that other groups have proven the sensitivity of PSA-NCAM hippocampal levels in AD mice to various treatments and even experiences [67]. In WT animals, PSA-NCAM up-regulation correlates with hippocampal-dependent learning [68]. Therefore, this particular marker reliably indicates the efficacy of the treatment strategy applied and monitors the improvements in hippocampal-dependent function.
In order to evaluate the treatment-associated changes in neuronal and dendritic density, we studied the brain expression patterns of MAP2. MAP2 belongs to a family of heat-stable microtubuleassociated proteins, which are responsible for polymerization, stabilization, and dynamics of the microtubule neuronal networks. Accordingly, MAP2 is vital for maintaining neuronal architecture, cell internal organization, cell division, and neuronal morphogenesis [69]. Of note, the levels of MAP2 are significantly diminished in the brains of AD patients [70]. Moreover, in vitro studies have shown that Aβ oligomers induce a time-dependent degradation of MAP2 in murine primary cerebral neurons [69]. Another in vitro study demonstrated the neuroprotective effect of curcumin, which up-regulates MAP2 expression in human neuroblastoma cells treated with Aβ oligomers [71]. Therefore, MAP2 levels in AD brains are potentially treatment-sensitive and can reflect the treatment efficacy.
It is noteworthy that 3×Tg mice exhibit an early neuronal loss [72] along with a significant reduction in the hippocampal spine density [16]. Previously, we applied Golgi staining and observed a significant increase in hippocampal spine density following norvaline treatment [16].
Here, we assessed the effects of the treatment on the neuronal and dendritic density in 3×Tg mice via quantitative immunohistochemistry with MAP2 antibody. Remarkably, the hippocampi of 3×Tg mice treated with norvaline showed significantly greater MAP2 signal than that of 3×Tg control mice (Fig. 3), These findings point to norvaline rescuing effects on neuronal and dendritic loss, which characterizes the development of memory deficits in 3×Tg mice, and in accord with our previously published data [16].
AD is accompanied by widespread neuroinflammation, and is characterized by chronic microglial activation and overproduction of proinflammatory cytokines [73]. Recent reports have suggested that proinflammatory cytokines, especially tumor necrosis factor-α (TNFα), negatively regulate adult mammal neurogenesis [74], whereas anti-inflammatory cytokines exert the opposite effect [73]. In our previous works, we have shown a significant effect of norvaline treatment upon the rate of microglial activation [16], and the levels of TNFα [15] in the brains of 3×Tg mice.
Accordingly, we suggest a supporting effect of norvaline upon adult neurogenesis through the reduction of neuroinflammation.
The small cytokine CCL11, which is produced by neurons in the brain, has been shown to be associated with immune response modulation and protection against neuroinflammation in rats [75]. More recent data strongly implicate the effect of CCL11 on mouse NPCs. Wang et al. (2017) utilized a rodent model of hypoxia-ischemia-induced brain damage to demonstrate that CCL11 promotes the migration and proliferation of NPCs [52]. Therefore, we reasoned that norvaline treatment would promote endogenous neurogenesis through neuroprotective factors such as antiinflammatory cytokines, particularly CCL11. We tested our hypothesis by analyzing CCL11 mRNA expression in the hippocampi of 3×Tg mice in relation to norvaline treatment and found a significant 79% treatment-associated increase. Accordingly, we speculate that CCL11 is at least partially responsible for the phenotype observed in the norvaline-treated mice. This relationship, however, is an assumption until proven by other assays, and further research is needed to shed light on the specific mechanisms of CCL11 induction by norvaline and its role in adult neurogenesis.
Norvaline is an efficient non-competitive arginase inhibitor [76], and this feature, is likely responsible for its neuroprotective properties. Of note, arginine is a common substrate for three enzymes present in several isoforms: arginase, NOS, and arginine decarboxylase (Fig. 5). These enzymes compete for mutual substrate reserves; thus, the overactivation of any one of them leads to the deprivation of others. AD development is associated with arginase overexpression at sites of β-amyloid deposition [12,16], which leads to brain arginine deprivation and also NOS and arginine decarboxylase substrate deficiency. When NOS is deprived of arginine, it undergoes uncoupling, which leads to considerable alterations in its mode of function; these changes reduce the production of NO and generate superoxide anion, which in turn, leads to severe oxidative stress (Fig. 5). omitted. In the AD brain, overactive arginase competes with NOS and ADC for the common substrate and reduces the bioavailability of arginine, which limits the production of agmatine and NO, and leads to NOS uncoupling and generation of superoxide anion. Overactivation of ornithine decarboxylase (ODC) leads to a surplus of downstream polyamine products, which can be neurotoxic [77]. Moreover, the gradual oxidation of polyamines by polyamine oxidase is associated with the generation of hydrogen peroxide and leads to oxidative stress [78].
NOS1 has been shown to be chiefly responsible for NO production in the brain and for regulation of vital physiological functions, including neurogenesis [11]. We demonstrated that norvaline upsurges the hippocampal levels of NOS1 [15] and NOS3 [16]; and therefore increases the brain NO content. We speculate that the neuroprotective effects of norvaline are mainly mediated by NO generation and the reduction of oxidative stress, which is a principal characteristic of AD [79].
The role of another arginine-processing enzyme in neurogenesis, arginine decarboxylase, has been recently determined. Arginine decarboxylase is responsible for the conversion of arginine into agmatine [80]. Accordingly, arginase inhibition-associated improvement in the substrate bioavailability leads to an elevation in agmatine brain levels (Fig. 5).
Agmatine has been shown to increase the proliferation rate of cultured hippocampal rat NPCs in vitro in a dose-dependent manner and also to induce hippocampal neurogenesis in chronically stressed mice in vivo [81]. A more recent study in rats showed that agmatine attenuates traumatic brain injury consequences via promoting neurogenesis and the inhibition of gliosis [82]. Another group proved that agmatine regulates NPCs proliferation and their fate determination in the SVZ [83]. These authors applied immunoblotting and staining to show that agmatine increases MAP2 levels, which supports our findings.
In summary, we have shown that long-term treatment with norvaline promotes NPC survival and differentiation in the hippocampi of 3×Tg mice. Our study offers new insights into the controlling of NPCs function by manipulating the NO microenvironment in the brain. It also provides additional evidence of arginase-targeting benefits in the treatment of AD.