Ontogenetic Changes in the Expression of the Lin28 Protein in the Rat Hypothalamic Tuberal Nuclei

The hypothalamus is a primary regulator of homeostasis, biological rhythms and adaptation to different environment factors. It also participates in the aging regulation. The expression of neurons containing Lin28 was studied by immunohistochemistry in male rats aged 2, 6, 12, and 24 months in the tuberal region of the rat hypothalamus. We have shown for the first time the presence of Lin28-immunoreactive (IR) neurons in the ventromedial nucleus (VMH) and their absence in the dorsomedial and arcuate nuclei in all studied animals. With aging, the percentage of Lin28-IR neurons increases from 37 ± 4.7 in 2-month-old rat until 76 ± 4.6 in 6-month-old and further decreases to 41 ± 7.3 in 12-month-old rat and 28 ± 5.5 in 24-month-old rats. Many VMH Lin28-IR neurons colocalized components of insulin signaling including mTOR, Raptor, PI3K and Akt. The percentage of Lin28/Akt-IR neurons was maximal in 6-month-old and 1-year-old rats compared to 2-month-old and 2-year-old animals. The proportion of Lin28/PI3K-IR neurons significantly increased from 77 ± 1.2 in 2-month-old rat until 99 ± 0.3 in 24-month-old rats and 96–99% of Lin28-IR neurons colocalized mTOR and mTORC1 component Raptor without statistically significant differences in all studied age groups. Thus, Lin28 expresses only in the VMH neurons of the tuberal nuclei of the hypothalamus and the Lin 28 expression changes during the development together with the components of PI3K-Akt-mTOR signaling.


Introduction
The highly conserved RNA-binding protein, Lin28, is involved in many biological processes, including development, control of onset of puberty and menopause, cell reprogramming, pluripotency and metabolism. Lin28 was initially discovered in Caenorhabditis elegans as a controller of time development and it has been found to selectively repress the expression of microRNAs, including those related to the Let-7 family [1,2]. Mammals produce two Lin28 paralogs, Lin28 (also known as Lin28a), and Lin28b. Lin28 and Lin28b are differentially localized in the cells: Lin28 is mainly found in cytoplasm, whereas Lin28b accumulates in the nucleus [3].
The Lin28/Let-7 axis is characterized by double negative feedback in the regulation of various biological functions. The level of let-7 is directly correlated with the expression levels of Lin28/Lin28b. Lin28 binds to the terminal loops of Let-7 precursors, leading to inhibition of processing and the induction of uridylation and degradation of the precursor. Lin28 is also a direct translational regulator: it selectively binds to a group of mRNAs and stimulates their translation [4,5].
Lin28 and Let-7 regulate the self-renewal and differentiation of stem cells. Lin28 or Lin28b is upregulated whereas Let-7a is downregulated in cancer and undifferentiated human and mouse embryonic stem cells relative to normal tissues. The Let-7 microRNA family members act as tumor suppressors by inhibiting expression of oncogenes and pluripotency factors including K-Ras, Cyclin D1, c-Myc, Cdc34, Hmga2, E2f2 and Lin28 [6,7].
The Lin28/Let-7 system is participated in the regulation of glucose metabolism and acts as a suppressor of multiple genes involved in the insulin signaling pathway [8][9][10]. Besides, Let 7 is involved in different pathways that regulate aging and aging-related diseases, and the level of Let-7 is elevated in aging tissues [11,12].
Lin28 is highly expressed in the hypothalamus in contrast with peripheral tissues. Expression Lin28 in the hypothalamus is affected by the metabolic state. Lin28 overexpression in the hypothalamus induced a significant improvement in the glucose metabolism and did not influence body weight [15].
The hypothalamus is a primary controller of homeostasis, biological rhythms and adaptation to different environment factors. It also participates in the aging regulation [16][17][18]. One of the manifestations of aging is the development of metabolic syndrome. Ventromedial (VMH), dorsomedial (DMH) and arcuate (ARH) hypothalamic nuclei participate in the regulation of metabolism and energy balance [19,20]. There are some data that neurons in the tuberal hypothalamic area control not only metabolic homeostasis but also lifespan [17,18]. In this case, expression of some markers changes in the hypothalamus of aged animals, for example calcium-binding proteins and neuronal NO synthase [21,22].
However, data on age-related changes in Lin28 expression are unavailable in the contemporary literature. Thus, the aim of the present study was to determine the location and percentage of Lin28-immunoreactive (IR) neurons as well as neurons co-expressing Lin28 with components of PI3K-Akt-mTOR signaling in the tuberal group of hypothalamic nuclei (ARH, DMH and VMH) in rats during aging.

Location and Percentage of Lin28-IR Neurons
We observed Lin28-IR neurons in the VMH of all studied rats. Immunoreactivity was located in the neuronal cytoplasm and proximal dendrites ( Figure 1). However, we did not find any Lin28-IR neurons in the DMH and ARH.  In the VMH, the percentage of Lin28-IR neurons significantly increased from 37 ± 4.7 in 2-month-old rat until 76 ± 4.6 in 6-month-old (p < 0.01) and significantly decreased later to 41 ± 7.3 in 1-year-old rat (p < 0.05 vs 6-month-old) and 28 ± 5.5 in 24-month-old animal (no statistically significant differences between 12-and 24-month-old, p > 0.05) ( Figure 2).

Discussion
We first studied expression of Lin28 in hypothalamic neurons located in the VMH, DMH and ARH in young, adult and aged rats. In the current study, we first observed Lin28-IR neurons only in the VMH in rats of different age groups from young until aged animals. ARH and DMH plays an important role in the metabolic regulation. Surprisingly, we did not observe Lin28-IR neurons in the above-mentioned nuclei. The percentage of Lin28-IR VMH neurons increases during the first 6 months of life and decreases in 1-yearold and aged 2-year-old rats. Low level of Lin28 in young rats was confirmed by Sangiao-

Discussion
We first studied expression of Lin28 in hypothalamic neurons located in the VMH, DMH and ARH in young, adult and aged rats. In the current study, we first observed Lin28-IR neurons only in the VMH in rats of different age groups from young until aged animals. ARH and DMH plays an important role in the metabolic regulation. Surprisingly, we did not observe Lin28-IR neurons in the above-mentioned nuclei. The percentage of Lin28-IR VMH neurons increases during the first 6 months of life and decreases in 1-year-old and aged 2-year-old rats. Low level of Lin28 in young rats was confirmed by Sangiao-Alvarellos et al. (2013), where Lin28 mRNAs displayed very high hypothalamic expression during the neonatal period but was minimal in the pubertal period [23].
We found that many VMH Lin28-IR neurons colocalized components of insulin signaling including mTOR, Raptor, PI3K and Akt1. However, patterns of expression of these components were different. The percentage of Lin28/Akt1-IR neurons had a peak in 6-month-old and 1-year-old rats. The proportion of Lin28/PI3K-IR neurons increased continuously and was maximal in aged 24-month-old rats. Nearly all Lin28-IR neurons colocalized mTOR and mTORC1 component Raptor without statistically significant differences in the all studied age groups from young until aged. From other studies, high levels of mTOR, Raptor and AkT were identified in the ARH and VMH [24]. From the literature data, Lin28 overexpression in the mouse VMH showed improved glucose tolerance and insulin sensitivity, while Lin28 downregulation during high fat diet was deleterious [8,15].
Lin28 upregulates components of the PI3K/AKT/mTOR pathway [8,11]. Akt phosphorylation level was increased in Lin28 VMH overexpressing mice and decreased in Lin28 knockdown mice [15]. In our previous data, the proportion of mTOR VMH neurons increased in the first six months of life and then decreased in 12-month-old and 24-month-old rats [25], similar to the pattern of changes of Lin28-IR VMH neurons, which we observed in the current work. Genetic inhibition of mTOR signaling can increase the lifespan of yeast, worms, and flies [26][27][28]. We can suggest that the downregulation of Lin28 and mTOR in VMH neurons may have a protective role during postnatal development and aging.
Lin28 inhibits the biogenesis of Let-7 miRNAs, which in turn repress Lin28 posttranscriptionally [11,29]. Members of the Let-7 miRNA family are involved in the regulation of glucose metabolism and act as suppressors of many genes involved in the insulin signaling pathway [30]. In previous studies, we found that, with aging, the expression of Let-7a microRNA in the nuclei of the medio-basal group of hypothalamic nuclei decreases, mainly in the DMH in males [30]. However, we did not find Lin28-IR neurons in the DMH. Nevertheless, Lin28 can affect various mRNA targets without altering Let-7 miRNA levels and Lin28-induced activation of Akt and mTOR may be not associated with changes in Let-7 expression [15,31].
Some recent data confirm the important role of the hypothalamus in the aging regulation. Cai group proposed microinflammation in the ventromedial hypothalamus as the main driver of aging [32,33]. Aging in mammals is accompanied by obesity and changes in hypothalamic neurons [16,34]. The VMH, which control the metabolic pathways, energy balance and peripheral circadian rhythms, may also participate in the programming of aging [35].

Animals
All experiments were performed in accordance with the Guidelines of the Russian Ministry of Health and EU Directive 2010/63/EU for animal experiments. Protocols were approved by the Ethics Committee of the Yaroslavl State Medical University. All necessary efforts have been made to reduce the number of animals and their suffering during the experiment. Experiments were performed on 2-month-old (weight 170-190 g), 6-month-old (weight 200-250 g), 12-month-old (weight 260-280 g), and 24-month-old (weight 330-350 g) male Wistar rats (5 animals in each group, 20 total). Animals were housed in an acclimatized room under 12 h light/dark cycle and a temperature of 22-24 • C with free access to food and water.

Immunohistochemistry
Immunohistochemistry was performed as previously described [21,22]. Briefly, rats were anesthetized with a lethal dose of urethane (3 g/kg, i.p.) and trans-cardially perfused within 10 min with 4% paraformaldehyde in phosphate-buffered saline (PBS, 0.01 M; pH 7.4). Brains were extracted and postfixed for 2 h in the same fixative at room temperature. After cryoprotecting in a solution of 30% sucrose in PBS (pH 7.4) at 4 • C, tissue was embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek Europe, the Netherlands), frozen and kept at −20 • C. Identification of VMH, DMH and ARH was carried out with the rat brain atlas [36]. The area of interest was identified by the presence of median eminence and the extension of the third ventricle (bregma from −2.4 mm to −3.2 mm). Brain tissue was sliced in the coronal plane with a cryotome (Shandon E, Thermo Fisher Scientific, Loughborough, UK). 14-µm-thick frozen sections were mounted on SuperFrost Plus (Menzel Gläser, Braunschweig, Germany) slides.

Image Acquisition
Slides were examined on Olympus BX43 fluorescence microscope (Olympus Europa, Hamburg, Germany) equipped with the following filter cubes: U-FBWA (Blue Excitation) and U-FGWA (Green Excitation VMH, DMH and ARH from each side were analyzed individually. Neuronal profiles with a clear identified nucleus were counted. To determine the percentage of IR cells, we counted the total number of neurons in the measured area and considered these as 100%. Three replicate measurements were performed for each capture region. The number of IR neurons was measured by ImageJ software (v. 1.53o 11 January 2022; http://imagej.nih. gov/ij/index.html).

Statistical Analysis
The statistical analysis was performed using Sigma Plot 12 software (Systat Software, Delaware, USA). The values are reported as the M ± SEM. Differences were assessed by one-way ANOVA with a post hoc Bonferroni's multiple comparison test. p values < 0.05 were considered statistically significant.

Conclusions
Our results indicate that aging is closely related to changes in the VMH neurochemistry. The molecular mechanisms leading to changes of Lin 28 and components of PI3K/AKT/mTOR signaling pathway in the hypothalamus need to be further explored.  Informed Consent Statement: Not applicable.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.