GADD34 Ablation Exacerbates Retinal Degeneration in P23H RHO Mice

The UPR is sustainably activated in degenerating retinas, leading to translational inhibition via p-eIF2α. Recent findings have demonstrated that ablation of growth arrest and DNA damage-inducible protein 34 (GADD34), a protein phosphatase 1 regulatory subunit permitting translational machinery operation through p-eIF2α elevation, does not impact the rate of translation in fast-degenerating rd16 mice. The current study aimed to validate whether P23H RHO mice degenerating at a slower pace manifest translational attenuation and whether GADD34 ablation impacts the rate of retinal degeneration via further suppression of retinal protein synthesis and apoptotic cell death. For this study, mice were examined with ERG and histological analyses. The molecular assessment was conducted in the naïve and LPS-challenged mice using Western blot and qRT-PCR analyses. Thus, this study demonstrates that the P23H RHO retinas manifest translational attenuation. However, GADD34 ablation resulted in a more prominent p-eIF2a increase without impacting the translation rate. GADD34 deficiency also led to a reduction in scotopic ERG amplitudes and an increased number of TUNEL-positive cells. Molecular analysis revealed that GADD34 deficiency reduces the expression of p-STAT3 and Il-6 while increasing the expression of Tnfa. Overall, the data indicate that GADD34 plays a multifunctional role. Under chronic UPR activation, GADD34 acts as a feedback player, dephosphorylating p-eIF2a, although this role does not seem to be critical. Additionally, GADD34 controls cytokine expression and STAT3 activation. Perhaps these molecular events are particularly important in controlling the pace of retinal degeneration.

Introduction
Retinal degenerative diseases comprise a wide heterogeneous group of retinal degenerative disorders, including inherited retinal degeneration (RD), affecting more than 200,000 Americans and individuals worldwide [1]. Retinitis pigmentosa (RP) is a group of rare eye dystrophies that, when inherited, lead to a gradual loss of rods and cones in the retina. Over 3000 mutations in more than 50 different genes result in a nonsyndromic form of RP, while over 1200 other pathogenic mutations cause syndromic forms, for example Usher and Bardet-Biedl syndromes [1]. Primarily, RP affects rod photoreceptors, leading to retinal dysfunction and cell death and causing the loss of night vision, followed by cone photoreceptor deterioration.
Mutations in rod photoreceptor genes encode aberrant proteins that lack the ability to properly fold. These synthesized misfolded proteins manifest compromised protein trafficking to cellular compartment destinations, such as the outer segments (OSs) of photoreceptors. Unable to transport to the OS and consistently misfolded, proteins accumulate in the endoplasmic reticulum (ER), thus compromising this organelle's homeostasis and activating a cellular program called the unfolded protein response (UPR). Mutant rhodopsin (RHO) is one of these proteins. The stress caused by misfolded proteins activates the protein kinase RNA-like endoplasmic reticulum kinase (UPR PERK) arm, promoting the phosphorylation (p) of eukaryotic initiation factor 2 alpha (eIF2α). In turn, p-eIF2α promotes a proadaptive signaling pathway-a temporal attenuation of global protein synthesis-whose major function is to cope with stress through adjustment of the translational rate. Overall, the PERK→p-eIF2α arm is also known as an integrative stress response (ISR).
Because retinal degenerative diseases are characterized by the consistent production of misfolded proteins, sustained ISR activation places a long-lasting hold on the recovery of protein synthesis in the deterioration of photoreceptor cells, leading to chronic translational attenuation. Recently, we investigated rd16 mice deficient in growth arrest and DNA damage-inducible Protein 34 (GADD34 −/− ). These mice expressed truncated CEP290, manifested chronic ISR activation, and developed severe RD rapidly, thus modeling Leber congenital amaurosis 10 and RP in humans. We demonstrated that GADD34-mediated manipulation with the p-eIF2α level in rd16 photoreceptors does not modify the translation rate and has no major impact on retinal function in mice with ciliopathies, although GADD34 ablation slowed down the rate of apoptotic cell death [2]. This is why we have become interested in understanding whether the GADD34-mediated elevation of p-eIF2α in retinas degenerating at a slower pace alters the progression of retinal dystrophy in mice.
Among several animal models of RP, there are P23H RHO mice with a relatively later onset and slower rate of RD. The proline to histidine at position 23 (P23H) RHO mutation accounts for about 12% of all autosomal dominant (ad) RP in North America [3,4]. It has been reported that human P23H RHO knock-in mice manifest accelerated photoreceptor cell degeneration and display decreased lengths of their OS [5]. In addition, the P23H protein is inadequately glycosylated with about 10% of WT RHO, suggesting that the mutation affects the consensus sequence for glycosylation at Asn 15 . Progressive retinal degeneration in these mice is obvious at Postnatal Day (P) 35. At this time point, a few rows of photoreceptor nuclei are missing and the amplitudes of the electroretinogram (ERG) recording (retinal function test) are significantly diminished as compared to the wild-type retina. These mice recapitulated the human retinopathy and showed rod photoreceptor functional deficit as compared to the cone system [5]. The protein structural study confirmed inefficiency in the signaling in P23H RHO, a perturbance in the P23H RHO protein dimerization, and a drastically altered mechanism of activation [6]. This pointed out that the ruptured Cys110-Cys187 disulfide bond in the extracellular loop 2 regions, the retinal binding site, of the protein structure is the source of extreme misfolding of P23H RHO through the entire receptor [6]. The P23H RHO mice demonstrated both activated ISR and an elevated p-eIF2α level [7].
The GADD34 protein has a multitude of other roles besides regulation of eIF2α activity including control of apoptosis. Moreover, both the inhibition and activation of apoptosis by GADD34 have been reported [8][9][10]. In addition to apoptosis, GADD34 is proposed to regulate STAT3. Thus, the study by Tanaka et al. proposed the GADD34 protein to regulate inflammation and host defense systems through the IL-6/STAT3 axis [11]. Moreover, both visual dysfunction and decreased RHO levels have been shown to correlate with increased STAT3 activation [12]. Therefore, in this study, we generated P23H RHO GADD3434 −/− mice lacking the PPP1R15A regulatory subunit of phosphatase holoenzyme protein phosphatase 1 (PP1)/GADD34 that dephosphorylates p-eIF2α to study its role in the RD of P23H RHO mice. This action was expected to elevate the p-eIF2α level in degenerating photoreceptors and to modulate the translation rate. We hypothesized that, similar to rd16 mice, the P23H RHO mice degenerating at a slower pace manifest the suppression of the translational rate, and the GADD34-mediated p-eIF2α elevation does not impact the rate of protein synthesis in their retinas; however, unlike rd16 mice, P23H RHO mice may require GADD34 to control apoptotic cell death. The data of this study indicate that the elevation of p-eIF2α in the P23H RHO GADD34 −/− retina does not lead to a more pronounced inhibition of protein synthesis, thus confirming the results obtained with rd16 mice and pointing out that, during chronically activated UPR, perhaps a mode other than the PERK→p-eIF2α mode controls the translational rate in degenerating photoreceptors [2,13]. In addition, the study revealed the anti-apoptotic role of GADD34 in P23H RHO GADD34 −/− photoreceptors with ongoing ISR. The p-PERK→p-eIF2α arm of UPR signaling is chronically upregulated in P23H RHO retinas starting from P19 [7]. Therefore, the p-eIF2α level was manipulated by ablating the PP1 regulatory subunit GADD34. Thus, it was found that a deficiency in GADD34 in the retina accelerated RD in P23H RHO mice. The a-and b-wave amplitudes of the scotopic ERG were registered, and it was found that GADD34 ablation reduced the a-wave amplitude as compared to the control, degenerating the retina by 28% ( Figure 1A,B, p < 0.05, n (number of animals) = 4-5), while no difference in the b-wave recording was observed between the experimental and control groups. Given the GADD34 ablation results in a decrease in the number of apoptotic cells in the rd16 retina, we then performed the detection of apoptosis by the TUNEL assay in P23H RHO GADD34 −/− retinas. Surprisingly, we learned that the decline in the photoreceptor-originated ERG waveform was in agreement with an increase in the number of apoptotic cell deaths in the P22 experimental degenerating retinas, suggesting that, most likely, GADD34 contributes to photoreceptor cell viability ( Figure 1C,D). Therefore, in the next step, the levels of chronically activated eIF2a and protein synthesis were identified in the P23H RHO retina.

GADD34 Ablation in P23H RHO Retinas Leads to an Increase in p-eIF2α and No Difference in the Rate of Protein Synthesis
First, it was found that GADD34 ablation results in dramatic upregulation of p-eIF2α in P23H RHO retinas by about twofold as compared to P23H RHO degenerating and wild-type retinas, respectively ( Figure 2A, p < 0.0001, n = 4). Therefore, in the next step, the translational rate in P23H RHO GADD34 −/− retinas was detected. First, it was observed that P23H RHO retinas with elevated p-eIF2α manifest a reduction in the rate of global protein synthesis ( Figure 2B,C, p < 0.0001, n = 4). No difference was found in the density of incorporated puromycin between P23H RHO and P23H RHO GADD34 −/− retinas, suggesting that, despite the dramatic increase in p-eIF2α, the level of protein synthesis is not inhibited further in P23H RHO GADD34 −/− mice. In agreement with these findings, the level of RHO, a major photoreceptor-specific protein, was found to have declined in both degenerating retinas ( Figure 2D, p < 0.0001 for P23H RHO, p < 0.0001 for P23H RHO GADD34 −/− , n = 4). Given an increase in the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive photoreceptor cells and a lack of changes in the translational rate and the RHO level, how the retinal cells increasingly die in the P23H RHO GADD34 −/− retinas remained unclear.

GADD34 Controls STAT3 Activation and Cytokine Expression in P23H RHO Degenerating Retinas
It is known that both visual dysfunction and decreased RHO levels have been shown to correlate with increased STAT3 activation [12]. Given a decline in RHO levels in the P23H RHO retinas ( Figure 2B), we became interested in the activation of the STAT3 transcriptional factor.
A significant over twofold elevation of p-STAT3 was observed in naïve P23H RHO retinas at P30 ( Figure 3A, p < 0.0001, n = 4) as compared to C57BL6 retinas, which correlated with the decrease in the RHO level depicted in Figure 2D. Interestingly, in P23H RHO GADD34 −/− mice, the p-STAT3 level was 18% lower compared to the P23H RHO control ( Figure 3A, p < 0.05, n = 4). These findings fueled our interest in whether the alteration in the p-STAT3 level correlates with cytokine expression in both RD mouse retinas. To compare the ability of both degenerating retinas to control cytokine expression, an inflammatory response was induced in the retinas by injecting LPS. First, it was found that the LPS-treated P23H RHO GADD34 −/− retinas manifest a 24% decline in p-STAT3 protein as compared to treated P23H RHO ( Figure 3B, p < 0.001, n = 5), and this fact was consistent with p-STAT3 activation in naïve retina. Second, an over threefold decrease in Il-6 and about a threefold increase in Tnfa mRNA expression in the LPS-treated P23H RHO retina-deficient GADD34 were found ( Figure 3C, p < 0.05 for both groups, n = 5), suggesting that cytokine expression is regulated by GADD34.      Given that P23H RHO GADD34 −/− retinas manifest an increase in the number of TUNEL-positive cells, which has been proven to be evidence of the apoptotic nature of cell death, ionized calcium-binding adaptor-molecule-1 (Iba1)-positive microglia and macrophages accumulated in the LPS-treated retinas were analyzed. Surprisingly, no difference was observed in the Iba1-positive cells when the two degenerating retinas were compared at 24 h ( Figure 3D,E), suggesting that GADD34 could regulate local cytokine expression.
Overall, the study demonstrated that GADD34's operation in degenerating retinas with ongoing UPR activation is necessary to control the progression of retinal pathogenesis in P23H RHO mice; GADD34 ablation in P23H RHO retinas results in a decrease in retinal function and an increase in apoptotic cell death.

Discussion
ISR is chronically activated in the P23H RHO retinas. Starting at P19, the p-eIF2α level was persistently upregulated [7]. These findings suggest that the rate of translation could be chronically attenuated in these retinas. Indeed, the rate of protein synthesis was found to be diminished in the P23H RHO retinas at P30. These results supported our previous findings with rapidly degenerating rd16 and rd10 retinas, both showing a decline in the rate of translation [14] and indicating that, overall, chronically upregulated ISR in the retina results in attenuation of global protein synthesis in varied animal models of RD.
The goal of the current study was to further test the hypothesis of whether manipulation with the p-eIF2α level could modify the rate of RD in mice deteriorating at a slower pace compared to rd16 and whether these changes are associated with further suppression of protein synthesis and an increase in apoptotic cell death.
GADD34, a protein phosphatase 1 regulatory subunit, plays a pivotal role in the regulation of eIF2α activity. Therefore, ablation of this protein is anticipated to provide sustained activation of eIF2α because PP1 phosphatase is no longer active. The data from this study showed an increase in p-eIF2α. Therefore, based on these findings, one can expect to observe the repression of protein synthesis on a larger scale. In contrast, the data of this study show that the further p-eIF2α increase does not inhibit global protein synthesis, thus supporting our published study demonstrating that GADD34 loss increases p-eIF2α, but has no impact on translation rates in RD [2]. This also implies that the PERK→p-eIF2α axis either does not play a major role in the regulation of protein synthesis or reaches a threshold of translational suppression, upon which further alteration in the p-eIF2α level does not affect the translational rate. For example, in the rd16 retinas, it was discovered that the mTOR/p-4EBP1/2 axis is a major mode of translational control [2]; therefore, a similar mode of translational regulation could operate in the P23H RHO retinas as well. The current and published studies of our group also indicate that, most likely, the inhibition of protein synthesis is not a cell defense mechanism in the retina by which deteriorating photoreceptors survive, but are the consequences of RD progression [13].
In the rd16 retinas, GADD34 ablation reduced the number of TUNEL-positive cells and the level of gliosis, although it did not result in a significant difference in scotopic ERG amplitudes [2]. In contrast, the P23H RHO GADD34 −/− retinas manifested robust TUNEL-positive staining as compared to the P23H RHO retinas. The results of this study also demonstrated that GADD34 is necessary for degenerating P23H RHO photoreceptors. GADD34 loss results in a small, but significant decline in scotopic a-wave ERG amplitudes. The increase in the number of TUNEL-positive cells supports these findings and provides evidence of more pronounced cell death in the P23H RHO GADD34 −/− retinas.
The primary functional role of GADD34 in cells with ER stress is to operate as a negative feedback loop during UPR activation, and its function allows for translation recovery through eIF2α dephosphorylation. Therefore, given the fact that the alteration in the p-eIF2α level does not affect the translational rate, how GADD34 controls retinal generation in mice was further explored in the current study. In addition to the PP1 regulatory function, GADD34 was shown to be required for normal cytokine production, both in vitro and in vivo. The cytokine production of IL-6 affected by GADD34 deletion/inactivation has been shown in multiple studies [11,15,16]. In this study, a reduction and increase in Il-6 and Tnfa mRNA expressions, respectively, were observed in degenerating retinas challenged with LPS.
Although the translational rate of the IL-6 cytokine in naïve P23H RHO was not accessed, it is possible that GADD34 could selectively control protein synthesis in the degenerating retinas. For example, Tanako et al. demonstrated a reduction of proinflammatory IL-6 cytokine production after LPS stimulation in GADD34 KO mice with colitis-associated cancer [11]. Additionally, an alternative mechanism-internal-ribosomeentry-site (IRES)-mediated translational control-could regulate IL-6 expression. Thus, it has been proposed that GADD34 promotes IRES activity to regulate protein expression in addition to the regulation of phosphorylation of eIF2a [17]. Interestingly, the regulatory IRES sequence has been reported for the IL-6 gene (hsa_ires_00025.1; http: //reprod.njmu.edu.cn/cgi-bin/iresbase/index.php). All these studies indicate that IL-6 expression could be regulated independently of the p-eIF2α→NF-kB→IL-6 axis, and the lack of GADD34 in P23H RHO LPS-treated mice could be responsible for the reduction of IL-6 cytokine in their retinas. Given the limitation of the current study, which did not provide the regulatory details of GADD34-mediated alteration of IL-6 and other individual protein expression escaping global translational attenuation, future experiments should shed light on the relationship between GADD34 and IL-6 in degenerating retinas.
In addition to IL-6, GADD34 controls Tnfa expression in P23H RHO retinas. It is known that preferential translation varies between stress. Under conditions of LPS-induced stress in P23H RHO GADD34 −/− retinas with enhanced p-eIF2a levels, TNF expression could occur in both p-eIFα-dependent and -independent manners. While studies demonstrated that TNFα expression is induced by the p-eIF2α→NF-kB→TNFa axis, other reports indicated that induction of p-eIF2α is not involved in the control of TNFα expression [18,19]. Thus, the research conducted in epithelial cells demonstrated that the application of guanobenz, a selective PP1 inhibitor, results in TNFα inhibition, and the mechanism of such activation is associated with upstream molecular events, such as interfering with the transforming growth factor (TGF)-β-activated kinase 1 (TAK1) complex [18]. Another study suggested that the PP1 regulatory subunit GADD34 can directly dephosphorylate IKK (Ser180/Ser181), returning the kinase to its default, inactive stage, thus regulating the downstream cytokine network [20]. This study implies that, in addition to the p-eIF2α→NF-kB→TNFa axis, TNFa could be regulated independently of the p-eIF2 level through GADD34-mediated phosphorylation of IKK. Therefore, the observed TNFα induction in the LPS-treated P23H RHO GADD34 −/− retinas with an increase in p-eIF2α could be a result of dual events: the activation of the p-eIF2α→NF-kB→TNFa axis and an increase in p-IKK-induced TNFα expression as a result of PP1 deficiency. Nevertheless, the TNFα reduction is beneficial to cone survival in mice with RD [21]. Therefore, a future study should be conducted to investigate the control mode of TNFα expression in the P23H RHO retina.
GADD34 mediates Il-6 expression. The IL-6 cytokine has been shown to regulate p-STAT3 [22,23]. The phosphorylation has been proposed to occur through JAK-mediated activation of STAT3 in response to stimulation by cytokines or growth factors [24]. We learned that p-STAT3 was also diminished in the LPS-treated retinas. Moreover, the p-STAT3 level declined in naïve P23H RHO retinas with GADD34 ablation. Altogether, these data demonstrate that GADD34 is upstream of STAT3 activation, which has been shown to act both as a regulator of retinal cell survival [25,26] and an inducer of retinal degenerative changes upon long-term activation [27].
Although the relationship between eIF2α and STAT3 has not been well investigated, it has been demonstrated that the activated STAT3 interacts with PERK and inhibits the phosphorylation of eIF2α [28]. In agreement with this study, we found a reduction of p-STAT3 in P23H RHO GADD34 −/− retinas with elevated p-eIF2α. Nevertheless, despite the observed reciprocal regulation of p-STAT3 and p-eIF2a, we believe that the major cause of p-eIF2α elevation is GADD34 deficiency. Therefore, to further assess the role of p-STAT3 in the RD of P23H RHO mice, one should trace the localization and phosphorylation patterns of the p-STAT3 protein in the course of RD.
Interestingly, the phosphorylation position seems to play an important role in the distribution of p-STAT3. Two major phosphorylation sites are known for STAT3: Y705 and S727. The phosphorylated STAT3 (S727) protein is primarily transported to mitochondria and is responsible for retinal ganglion cell (RGC) axon regeneration upon stress [29]; p-STAT3 (Y705) is localized in the nucleus and found in the retinas of animal models of RD. For example, a recent study conducted in the Gross lab showed Q344X RHO knock-in mouse retinas manifesting sustained p-STAT (Y705) upregulation during RD confined to the nuclei of Müller cells in the retinal inner nuclear layer (INL) [30]. Other retinal degenerative models, transgenic RHO P347S and Prp2rds +/− (perpherin-2, retinal degeneration slow) mice, manifest similar p-STAT3 (Y705) levels in Müller glia, in addition to a weak immunoreactivity for p-STAT3 in photoreceptors [26]. In contrast, a group led by John Ash identified p-STAT3 (Y705) in photoreceptors treated with leukemia inhibitory factor [31]. It needs to be emphasized that both conducted studies are in agreement with the prosurvival role of STAT3 activation in degenerating retinas [26,31]. These studies, together with the previous findings, indicate that GADD34 may play a local role in degenerating retinas. As evidence, GADD34 regulates Il-6 expression and p-STAT3 activation in the retina. Deficiency in GADD34 leads to a reduction in p-STAT3 and the accelerated rate of photoreceptor cell death in P23H RHO retinas.
In conclusion, it is hypothesized that GADD34 plays a multifunctional role in the degenerating retina. On the one hand, under chronic UPR activation, GADD34 acts as a feedback player by dephosphorylating p-eIF2α, although this role does not seem to be critical due to the lack of further translational repression in degenerating retinas. On the other hand, GADD34 controls the IL-6/STAT3 arm and STAT3 activation, a lack of which compromises photoreceptor cell viability, leading to a decrease in the scotopic a-wave amplitude and an increase in apoptotic cell death in degenerating retinas. These late events may control the pace of RD in the P23H RHO retinas.

Animals
The P23H RHO and C57BL6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The GADD34 −/− mice were produced as previously described [2]. Then, the P23H RHO mice were crossed with GADD34 −/− and housed in the UAB animal core facility with a 12 h light/dark cycle with free access to a standard diet and water. Retinas from control and experimental mice of both sexes were used at Postnatal Days (P) 22 and 30. Mice were euthanized by CO 2 asphyxiation followed by cervical dislocation. All animal experiments followed a protocol (IACUC#21044) approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee (IACUC) and conformed to guidelines from the Association of Research in Vision Science and Ophthalmology.

Electroretinography
To conduct the scotopic electroretinography (ERG) recording, the LKC BigShot ERG (Gaithersburg, MD, USA) instrument was used. To that end, the mice were dark-adapted overnight and then anesthetized with an intraperitoneal injection of ketamine/xylazine on the basis of their weight. Topical 2.5% phenylephrine (Paragon BioTeck, Inc., 42702-102-15, Portland, OR, USA) and Gonak, 2.5% sterile hypromellose ophthalmic demulcent solution (AKORN, Lake Forest, IL, USA), were applied to the corneal surfaces of the mice. Then, we placed a monopolar contact loop on the surface of the cornea to register the retinal aand b-wave ERG amplitudes. The reference electrodes were placed under the scalp and in the tail. Then, the mice were exposed to five flashes of 0.025 cd*s/m 2 , 2.5 cd*s/m 2 , 7.91 cd*s/m 2 , and 25 cd*s/m 2 light intensities with intervals of 45 s. Their ERGs were recorded simultaneously. The a-and b-waveforms were analyzed using the LKC EM software (Gaithersburg, MD, USA).

Histology and Terminal Deoxynucleotidyl Transferase dUTP nick-End Labeling Assay
The eyes of the mice were enucleated at P22 and placed in 4% paraformaldehyde (PFA) (Santa Cruz Biotechnology Inc., sc-281692, Dallas, TX, USA). A 33G needle was used to create a small hole for eye tissue fixation in 4% PFA for 1 h. Then, we washed the eyes with phosphate buffered saline (PBS) (Gibco, 10010-023, Paisley, UK) and immersed them in 30% sucrose overnight. The eyes were further examined for cryopreservation in an optimal cutting temperature (OCT) compound (VWR: 25608-930). The 12 µM retinal cryosections were performed using a sectioning system (Leica CM 1510S; Leica, Buffalo Grove, IL, USA).
To detect apoptotic cell death, TUNEL staining (Click-iT Plus TUNEL assay, Ther-moFisher Scientific, C10617, Waltham, MA, USA) was performed according to the protocol of the manufacturer's procedure on retinal sections from mice at P22. Whole eyes were removed and fixed in 4% PFA for 20 min on ice, and a needle (33G) was inserted at the limbus to create a small hole, following incubation for 2 h. Then, the fixed eyes were placed in 30% sucrose in PBS overnight at 4 • C. Eyes were cryopreserved in Tissue-Tek OCT compound (Sakura Fintek USA Inc., 4583, Torrance, CA, USA) and kept at −80 • C until sectioning. The samples were cut into 12 µm-thick sections on a CM 1510S cryostat (Leica, Deer Park, IL, USA).

LPS Experiment
The P30 mice were injected with 10 mL/kg (stock solution of 1 mg/mL) of lipopolysaccharide (LPS) intraperitoneally at P30, as previously described [32]. After 6 and 24 h, the mice were sacrificed, and their eyeballs were enucleated for Western blot and qRT-PCR analyses, respectively. At 24 h post-injection, immunostaining to identify Iba1-positive cells was also performed. For Iba1-positive microglial cell counting, the cryosections were rinsed with PBS and then blocked in 5% normal donkey serum and 0.3% Triton X-100 in PBS for 1 h at room temperature. The retinal cryosections were then incubated in the primary anti-IBA-1 Antibody (Wako Chemicals, 019-19741, Osaka, Japan). The secondary Alexa Fluor 555 or 488 anti-rabbit antibodies (ThermoFisher Scientific, A31572; A21206, Eugene, OR, USA) diluted in PBS (1:500) were applied for 2 h at room temperature. Nuclei were visualized by counterstaining with DAPI in a fluorescence mounting medium (Southern Biotech, 0100-20, Birmingham, AL, USA), and images were acquired with a Keyence BZ-X800 Fluorescence Microscope (Itasca, IL, USA). Investigators blinded to the results counted the number of Iba1-positive cells in the retinal sections. Fluorescent images were taken, and the Iba1-positive cells were calculated in a vision field of 200 µm 2 .

Analysis of Nascent Protein Synthesis
The SUrface SEnsing of Translation (SUnSET) method has been described previously [33]. Briefly, the mice were injected with puromycin (puromycin dihydrochloride; Santa Cruz Biotechnology, CAS 58-58-2, Dallas, TX, USA) intraperitoneally at a dosage of 0.04 µmol/g body mass. Then, mice were sacrificed, and their retinas were harvested to prepare protein extracts with RIPA buffer (Cell Signaling, 98065, Danvers, MA, USA). Both retinas from each mice were combined into one sample. Total protein (40 µg per well) was electrophoresed on a 4-12% SDS-polyacrylamide gradient gel (Bio-Rad, Hercules, CA, USA) and blotted onto a PVDF membrane. The antibodies specific to puromycin (mouse, MABE343, Millipore Sigma, St. Luis, MO, USA) were applied to incubate the membranes. A secondary antibody specific to IgG2a (goat antimouse peroxidase affinipure IgG, Fcγ Subclass 2a Specific: 115-035-206, Jackson Immuno Research Laboratories Inc.; West Grove, PA, USA) was reused to detect the bands. After scanning the density of the puromycin-incorporated bands using a LI-COR Odyssey XF imager (Lincoln, NE, USA), the membranes were stained with Coomassie Brilliant Blue R-250 Stain (Bio-Rad, 1610436, Hercules, CA, USA) for their normalization. The relative densities of the entire lanes of Coomassie-stained proteins and of incorporated puromycin were calculated using the ImageJ software and used to quantify the rate of protein synthesis.

Real-Time Quantitative PCR
The retinas were dissected and frozen in liquid nitrogen. Samples were stored at −80 • C until processing. RNA was isolated from the retinas using TRIzol (ThermoFisher Scientific, 15596026, Waltham, MA, USA). Genomic DNA was removed using ezDNAse cDNA was synthesized from 500 µg of RNA using SuperScript IV VILO Master Mix (ThermoFisher Scientific, 11766050, Waltham, MA, USA). A cDNA synthesis reaction mixture containing no reverse transcriptase was used as a negative control in subsequent amplifications to confirm the absence of genomic DNA contamination. The following TaqMan primers (ThermoFisher Scientific, Waltham, MA, USA) were used: Il6 (Mm00446190_m1), Tnfα (Mm99999068_m1), GAPDH (Mm99999915_g1). Quantitative RT-qPCR was performed in duplicate using iTaq Universal Probe Supermix (Bio-Rad, 1725131, Hercules, CA, USA) on a QuantStudio 3, 96 well plate Real time PCR machine (ThermoFisher Scientific, A28131, Waltham, MA, USA). Gene expression was calculated using the 2 −∆∆CT method and normalized against the expression of GAPDH.

Statistics
The Student t-test was used to compare two groups, and ordinary one-way ANOVA (Tukey's test) and two-way ANOVA (Sidak's test) with multiple comparisons were carried out to compare three groups of animals. All statistics were calculated using GraphPad Prism 9 software (San Diego, CA, USA).

Conclusions
In conclusion, it was hypothesized that GADD34 ablation exacerbates retinal degeneration in P23H RHO mice. We found that this protein plays a multifunctional role in degenerating retina. On the one hand, under chronic UPR activation, GADD34 acts as a feedback player by dephosphorylating p-eIF2α, although this role does not seem to be critical due to the lack of further translational repression in degenerating retinas. On the other hand, GADD34 controls the IL6/STAT3 arm and STAT3 activation, a lack of which compromises photoreceptor cell viability, leading to a decrease in the scotopic a-wave amplitude and an increase in apoptotic cell death in degenerating retinas. These late events may control the pace of RD and apoptotic cell death in the P23H RHO retinas.


The Edible Plant Crithmum maritimum Shows Nutraceutical Properties by Targeting Energy Metabolism in Hepatic Cancer

In the past few years, evidence has supported the role of plants as a valuable tool for the development of promising therapeutic support options for many diseases, including cancer. We recently discovered that the edible wild plant Crithmum maritimum L. effectively inhibits the growth of hepatocellular carcinoma (HCC) cells and we provide insights into the biological mechanisms involved. Here, we aimed to characterize the effect of ethyl acetate extract of Crithmum maritimum on the bioenergetic phenotype of HCC cells and if this is associated with the anti-tumour effect we previously described. Results show that Crithmum maritimum significantly increases cellular respiration and reduces lactic fermentation in HCC cells, and that this reduction of the fermentative glycolytic phenotype is linked to inhibition of HCC growth. These data provide new preclinical evidence supporting the role of Crithmum maritimum L. as a nutraceutical option to expand the therapeutic opportunities in the management of HCC. Supplementary Information The online version contains supplementary material available at 10.1007/s11130-022-00986-z.

Introduction
The utilisation of plant extracts and plant-derived products for therapeutic purposes is now gaining new importance for many pathological affections, including cancer [1]. Hepatocellular carcinoma (HCC) is nowadays the sixth leading cause of tumour-related deaths worldwide and expected as the third cause in Western countries by 2030 [2]. HCC management mainly relies on surgery, while the pharmacological approach to tyrosine-kinase inhibitors, such as sorafenib [3], is often combined with immunotherapy drugs. Such protocols have many adverse effects and cannot be tolerated in the long-range. Therefore, a novel effective, and better-tolerated therapeutic options for HCC are extremely needed.
Crithmum maritimum is known for a long time as an edible plant with general beneficial health effects, with antioxidant and antimicrobial properties [4]. Additional details on botanical, geographical and traditional medicinal and food uses are reported in Supplementary Material. We previously demonstrated that Crithmum maritimum ethyl acetate extract reduced HCC cell growth with low toxicity [5] and changed the metabolic profile, decreasing the level of choline and phosphocholine, lactate, amino acids, and cholesterol, directly involved in the control of cell growth [6] (Fig. S2b). This suggests that the cytostatic action exerted by Crithmum maritimum is at least in part due to the modulation of cellular metabolism. Also, Crithmum maritimum reduced the level of monounsaturated fatty acids (MUFA) and increased that of polyunsaturated fatty acids (PUFA) (Fig. S2b) [6]. We also provided preclinical evidence that Crithmum maritimum can be used in combination with sorafenib to reduce its dose and toxicity ( Figure S3) [7].
We have been the first to describe and characterize the activity of Crithmum maritimum in inhibiting HCC growth [5], associating this effect with a complex multi-target action on HCC cell metabolism [6]. Here, we demonstrated that Crithmum maritimum changes the bioenergetic phenotype of HCC cells, activating oxidative phosphorylation (OXPHOS) and decreasing lactic fermentation. These observations support the view that activation of aerobic oxidative metabolism is associated with a reduction of tumour growth.

Materials and Methods
The material and method section is reported in the Supplementary Material.

Results and Discussion
We previously reported that Crithmum maritimum ethyl acetate extract effectively inhibits HCC cell growth [5] and determines a deep remodelling of the HCC cell metabolic profile [6]. Here, we aimed to characterise the role of Crithmum maritimum ethyl acetate extract on HCC cell bioenergetic phenotype and if this is associated with its anti-tumour effect [5,6]. We assessed cellular respiration by employing a polarographic oxygraphy approach and determined intracellular lactate production and lactate dehydrogenase (LDH) activity. Figure 1a-c demonstrates the cytostatic effect after a 24 h treatment with Crithmum maritimum ethyl acetate extract in two HCC cell lines, HepG2 and Huh7. A 24 h treatment in the same conditions also determined a significant increase in basal respiration, ATP-dependent respiration and respiratory reserve, in both cell lines (Fig. 1b-d). Representative oxygraphy graphs are shown in Figure s4. More details about oxygraphic measurement can be found in Supplementary Material. The increased sensitivity of HCC cells to ATP synthase inhibitor oligomycin observed in oxygraphy measurements was confirmed by assessing oligomycin-induced cell toxicity after a 24 h pre-treatment with Crithmum maritimum (Fig. 1e-f). This corroborates the effects of Crithmum maritimum in activating ATP-dependent respiration. Figure 1b-d outlines the overall effect of Crithmum maritimum ethyl acetate extract on the activation of cellular respiration. Finally, Fig. 1g-h shows a significant decrease in intracellular lactate production and LDH activity after 24 h treatment with Crithmum maritimum. In summary, this supports the view that Crithmum maritimum shifts the bioenergetic phenotype of HCC cells from anaerobic glycolysis towards OXPHOS and this results in an inhibition of tumour growth.

Conclusions
Based on our findings, we propose Crithmum maritimum as an effective choice for the development of new nutraceutical strategies in the treatment of HCC, aimed to support conventional HCC pharmacological therapies and to reduce side effects. Future research activity will be directed to the preparation of formulations suitable for human clinical trials to conduct case-control studies on HCC patients, combining appropriate extract preparations with standard therapies. Overall, we believe that this edible plant owns the potentiality to open the way for effective therapeutic options for HCC as well as for liver metabolic diseases, and possibly to other types of cancers.

Declarations
Ethics Approval Not applicable.

Consent for Publication
All authors have given their consent for publication.

Conflict of Interest
Authors have no potential conflicts of interest to disclose.
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Chromosome-level Assembly, Dosage Compensation and Sex-biased Gene Expression in the Small Brown Planthopper, Laodelphax striatellus

Abstract In insects, sex chromosome differentiation often results in unequal gene dosages between sexes. Dosage compensation mechanisms evolve to balance gene expression, but the degree and mechanism of regulation often vary by insect species. In hemipteran species, the small brown planthopper (SBPH), Laodelphax striatellus, is an injurious crop pest, with a sex chromosome type XX in females and XO in males. This species offers the opportunity to study dosage compensation and sex-biased gene expression. In this study, we generated a chromosome-level genome of SBPH using Oxford Nanopore Technologies and high-throughput chromatin conformation capture (Hi-C) technology. We also sequenced RNA-seq data from 16 tissue samples to annotate the genome and analyze gene dosage compensation. We finally obtained a 510.2 megabases (Mb) genome with 99.12% of the scaffolds anchored on 15 chromosomes (14 autosomes and 1 X chromosome) and annotated 16,160 protein-coding genes based on full-length cDNA sequencing data. Furthermore, we found complete dosage compensation in all L. striatellus somatic tissues, but lack of dosage compensation in gonad tissue testis. We also found that female-biased genes were significantly enriched on the X chromosome in all tissues, whereas male-biased genes in gonad tissues were enriched on autosomes. This study not only provides a high-quality genome assembly but also lays a foundation for a better understanding of the sexual regulatory network in hemipteran insects.

Introduction
Sex chromosome differentiation is a common way to determine sexes in nature (e.g., two basic sex chromosome types: XX females and XY males or ZW females and ZZ males). The sex chromosomes evolve from a normal chromosome pair that carries sex-determining genes (Ohno 1967). The region containing these sex-determining genes will become recombination suppressed, which contributes to subsequent genetic degeneration of sex chromosome regions (e.g., massive gene loss or even complete loss of the Y or W chromosome (Bergero and Charlesworth 2009). This degeneration triggers the problem of unequal gene dosage on sex chromosomes, which could be lethal or deleterious. To mitigate the impact, the mechanism of dosage compensation evolves to balance gene expression on sex chromosomes between females and males and between sex chromosomes and autosomes (Disteche 2012;Graves 2016). Studies involving different lineages have demonstrated that the extent of dosage compensation and the precise molecular mechanisms vary among species. In therian mammals, one entire X chromosome is randomly inactivated in somatic cells of females by epigenetic silencing to balance the dosage difference (Lyon 1961;Gartler and Riggs 1983). In Drosophila, dosage compensation is regulated by upregulating the X chromosome in males (Larschan et al. 2011). In birds, reptiles and fish, dosage compensation is partial and gene specific (Ellegren et al. 2007;Vicoso et al. 2013;Shao et al. 2014).
Insects are the most speciose group in the animal kingdom. At present, limited studies regarding dosage compensation have been conducted in certain species of Diptera (Nozawa et al. 2014;Vensko and Stone 2015), Lepidoptera (Catalán et al. 2018;Ranz et al. 2021), Hemiptera (Pal and Vicoso 2015;Richard et al. 2017), Orthoptera (Chauhan et al. 2021), Strepsiptera (Mahajan and Bachtrog 2015), and Coleoptera (Prince et al. 2010), whereas it remains unclear in a tremendous number of other species. Based on existing results, the degree of dosage compensation and regulation mechanisms vary among insect species and even different tissues (Nozawa et al. 2014;Ranz et al. 2021), developmental stages (Rose et al. 2016), and time periods of segment added to X chromosomes (Mahajan and Bachtrog 2015;Gu et al. 2019). Hemiptera is an ideal clade to study the evolution of sex chromosomes. In this group, many species show degenerated Y chromosomes with XY or XO sex determination systems. The dosage compensation in pea aphid, Acyrthosiphon pisum (Richard et al. 2017), and three true bugs (Pal and Vicoso 2015) have been analyzed, all show the existence of dosage compensation, but these studies only analyze whole body samples, which could not represent the dosage compensation state among different tissues. In addition, X-chromosome masculinization has been found in pea aphid, whereas feminization has been found in other three true bugs. We still know little about dosage compensation in rice planthoppers. The small brown planthopper, Laodelphax striatellus (Fallén) (Delphacidae, Hemiptera), is an important agricultural pest that damages a variety of cereal crops, including rice, wheat, maize, oats, tall oat grass, and barley, as well as transmits several important rice viruses. Its distribution widely covers Asia, Europe and some areas of Oceania and Africa. From 2006 to 2015, rice planthoppers have caused an average of ∼13.1 million tons of economic and yield losses each year in China (Liu et al. 2016).
In recent years, genome assembly combined with RNA-seq data has revealed the state of dosage compensation in many species. The previous draft genome version of SBPH has been assembled by high-depth of Illumina short reads and use low-depth of Pacific Biosciences (PacBio) data to fill the gaps. The scaffold-level genome has a contig N50 of 118 kilobases (kb) and a scaffold N50 of 1.08 Mb (Zhu et al. 2017). Later, this draft genome has been assembled to chromosomal level with Hi-C technology (Ma et al. 2021). However, nearly 20.4% of the genome regions have failed to locate on any chromosome, which may be due to the short length of contig N50 and the lack of restriction sites in many short contigs. The third-generation sequencers represented by PacBio and Oxford Nanopore Technologies (ONT) MinION generate longer reads, reaching tens of kilobases (Laver et al. 2015), greatly improving genome assembly quality. For example, application of ONT and Hi-C data has increased scaffold N50 of Illumina sequence assembly from 356.6 kb to 69.96 Mb for Nilaparvata lugens (Ye et al. 2021).
In this study, we assembled the chromosome-level genome of SBPH by combining ONT sequences and Hi-C technology to improve genome quality. By analyzing the transcriptome gene expression, we tested the level of dosage compensation and characterized how sex-biased genes were distributed across chromosomes in different tissues of SBPH.

Chromosome-level Assembly of the L. striatellus Genome
We sequenced the male genome of L. striatellus using four 1D flow cells by ONT 1D. After quality control, a total of 106.89 gigabases (Gb; ∼200X) of clean data was retained, including 16,381,943 subreads with a read N50 length of 21,754 bases and an average read length of 6,525 bases (supplementary table S1, Supplementary Material online). We also sequenced 34 Gb (∼68X) of clean Illumina data of male L. striatellus to estimate genome size and polish the genome assembly. We applied Hi-C data (supplementary table S3, Supplementary Material online) to construct haploid contigs for chromosome-level assembly. The length of the final chromosome-level genome was 510,210,580 bp and the scaffold N50 was 34,959,101 bp (table 1). The genome assembly included 15 chromosome-level scaffolds (lengths ranged from 13,928,342 to 59,587,101 bases, accounting for 99.13% of the total genome), and additional 297 small scaffolds (4,460,860 bases in total, accounting for 0.87% of the genome; fig. 1A). The GC content of the male L. striatellus genome was 34.52% ( fig. 2B), which was very close to the previous version's 34.54% (Zhu et al. 2017). To assess the completeness of the genome assembly, benchmarking universal singlecopy orthologs (BUSCOs) of Eukaryota (n = 255), Arthropoda (n = 1,013), Hemiptera (n = 2,510), and Insecta (n = 1,367) were used to align against the genome, and the completeness rate ranged from 92.2% to 95.9% ( fig. 3A). These results demonstrated the high quality of our genome assembly. Furthermore, we identified a total of 207,334 simple sequence repeats (SSRs) from the whole genome (supplementary table S4  Genome Biol. Evol. 14(11) https://doi.org/10.1093/gbe/evac160 Advance Access publication 1 November 2022 GBE X Chromosome Identification The karyotype of L. striatellus consists of 15 haploid chromosomes (14A + 1X; Zhu et al. 2017), which is the same number as our Hi-C chromosome-level scaffolds (figs. 1A and 2A). To identify the X chromosome, ∼34 Gb of males and 33 Gb of females Illumina paired-end DNA short sequences were mapped against the genome to estimate the coverage of the mapped reads. We then calculated male to female coverage ratio in a 100-kb sliding window across chromosomes. The method of sex determination in L. striatellus is that females have two X chromosomes (XX) and males have only one X chromosome (XO), so the sequence read coverage of the X chromosome in males should be half that of females. In our results, only one chromosome, the X chromosome, was observed with a male to female coverage of about 0.5, while the ratio of remaining autosomes fluctuated close to 1 ( fig. 1B).
Genome Annotation-based on ONT Long-read and Illumina Short-read cDNA Sequencing To obtain accurate gene structure annotation, we sequenced both Illumina short-read and ONT long-read cDNA data to perform genome annotation. Illumina data were obtained from six independent libraries of L. striatellus body tissues, including carcass, fat body, gut, ovary, testis, and salivary glands. After quality control, ∼97.64 Gb of clean data was retained, containing 650,918,608 sequences of 150 bp. Then, these short reads were assembled into 154,426 transcripts by Trinity. ONT data were generated from four independent libraries of L. striatellus at different developmental stages, including eggs, nymphs, adult males, and adult females. A total of 8,322,589,622 bases were obtained, consisting of 6,821,102 reads, with an average read length of 1,220 bp. The reads were further classified into 155,475 full-length consensus isoforms with a read length N50 of 3,003 bp (supplementary table S6, Supplementary Material online).
We finally annotated 16,160 protein-coding genes across the genome of L. striatellus (supplementary table  S7 (supplementary  table S8, Supplementary Material online). Overall, the BUSCO completeness and the high annotation rate indicated that our gene annotation was accurate and reliable.

Dosage Compensation of L. striatellus
To investigate whether the degree of dosage compensation differs in different L. striatellus body tissues, we obtained Illumina RNA-sequencing data from gonad tissues (female ovaries and male testes) and used publicly available somatic transcriptome data from legs, antennae, and heads (Li et al. 2021). To analyze dosage compensation, we compared gene expression levels between females (XX) and males (X) and between the X chromosome and autosomes within each sex.
We first examined the expression levels of the X chromosome versus autosomes in males (X:AA) and females (XX: AA; fig. 4A). We found that expression levels of autosomes were significantly higher than X chromosomes in all male tissues ( Genome Biol. Evol. 14(11) https://doi.org/10.1093/gbe/evac160 Advance Access publication 1 November 2022 and somatic tissues showed 3-5% higher median expression in autosomes compared with that of the X chromosome (supplementary table S11, Supplementary Material online). In contrast, there were no significant differences between X chromosomes and autosomes in female tissues (except legs; fig. 4A, supplementary table S10, Supplementary Material online; Wilcoxon P-values: head, 0.086; antenna, 0.1328; leg, 0.0442; gonad, 0.19). We also investigated the X:A expression ratio of females and males median chromosome expression. We found that in males, the X:A ratio ranged from 0.952 to 0.976, and in females, it ranged from 0.965 to 1.002 in somatic tissues. For gonad tissues, X:A ratio was 0.552 in testis and 1.031 in ovary ( fig. 5A, supplementary table S10, Supplementary Material online).
In species with dosage compensation, expression should be balanced between one X (X) in males and two X (XX) in females. Therefore, we further compared differences in the X-chromosome expression levels between males and females. We found no significant differences in the X-chromosome expression levels between males and females in all somatic tissues ( fig. 4B; supplementary  Taken together, these results suggest the existence of complete dosage compensation in the somatic tissue of L. striatellus. In gonad tissue, however, the male testis lacks dosage compensation.

Genomic Distribution of sex-biased Genes
Sex-biased genes are often nonrandomly located on chromosomes in many species, with sex chromosomes usually showing a pattern of masculinization or feminization. To analyze whether sex-biased genes have skewed distribution in L. striatellus, we used our gene structure annotation and X chromosomes (X). The horizontal lines on the top and bottom of the box represent the quartiles, the horizontal line in the middle represents the median, and the two whiskers represent the 1.5X interquartile range. Asterisks indicate significant differences between females and males, and significance was determined by Wilcoxon test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant. GBE results to infer the chromosomal locations of sex-biased and unbiased genes in somatic and gonad tissues. We observed a significantly higher proportion of female-biased genes on X chromosomes than autosomes in all tissues of L. striatellus, suggesting an enrichment of female-biased genes on the X chromosome ( fig. 6A; supplementary table S12, Supplementary Material online). On the other hand, we found a significantly higher proportion of male-biased genes in autosomes compared with X chromosome in gonad tissue. In somatic tissues, the proportion of male-biased genes was higher in autosomes than on the X chromosome, although this difference was not significant ( fig. 6B; supplementary table S12, Supplementary Material online). Sexual antagonism and possible lack of dosage compensation in the testis may drive X-chromosome enrichment of female-biased genes and autosome enrichment of malebiased genes (see Discussion).

Discussion
The previous two versions of the L. striatellus genome either have a relatively short scaffold N50 (Zhu et al. 2017) or leave ∼20% of scaffolds unplaced on chromosomes (Ma et al. 2021). Here, we improved the genome quality of L. striatellus. The new assembly demonstrated higher contiguity than previous versions at both contig and scaffold levels, with contig N50 increased from 71 to 1,362 kb and scaffold N50 increased from 29.24 to 34.96 Mb (table 1). In addition, most scaffolds (99.12%) were anchored to chromosomes, with a lower proportion of ambiguous nucleotides FIG. 5.-Expression ratios between chromosomes and between sexes. (A) Median X-to-autosomal expression ratio (X:A) in females and males. Dots represent the median X-chromosomal expression level log 2 FPKM versus median autosomal expression level (X/A expression ratio) in females (F) and males (M), respectively. (B) Boxplots show the distribution of the median female to male expression ratios on autosomes and X chromosomes. The horizontal lines above and below the box represent the quartiles, the horizontal line in the middle represents the median, and the two whiskers represent the 1.5X interquartile range. Asterisks indicate significant differences between autosomes and X chromosomes, and significance was determined by Wilcoxon test. ***P < 0.001, ns, not significant. 6.-Chromosomal enrichment of female-and male-biased genes. Asterisks indicate significant differences in the proportion of sex-biased genes on autosomes and on X chromosomes. Significance was determined by Fisher's exact test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant. (A) Female-biased gene enrichment analysis. (B) Male-biased gene enrichment analysis.

FIG.
Genome Biol. Evol. 14(11) https://doi.org/10.1093/gbe/evac160 Advance Access publication 1 November 2022 (0.04%). Furthermore, BUSCO analysis showed that 95.9% of the complete Hemiptera orthologous genes were identified in this genome. Thus, the quality of our present L. striatellus genome assembly is improved as it is more contiguous, accurate, and complete.
We found that in somatic tissues of L. striatellus, the degree of sex chromosome dosage compensation in males was complete. Gu and Walters (2017) argued that sex chromosome dosage compensation should be limited to that heterogametic X-linked gene expression equal to ancestral level of the proto-X, and the equality of sex-linked expression between the sexes was termed as dosage balance. In L. striatellus, we found equal level of X chromosome expression between males and females, and no difference in expression between X chromosome and autosomes in homogametic females. Thus, complete compensation with balance existed in L. striatellus somatic tissues. Although we found that the X-chromosome expression levels were significantly lower than autosomes ( fig. 4A), and the median X:A ratios were close to but still <1 in male somatic tissues ( fig. 5A). First of all, some genes may escape dosage compensation. It has been reported that Drosophila's ability to bear a deficiency in gene dose varied depending on the gene (Lee et al. 2016). Thus, lack of dosage compensation in some genes may cause subtle or even no effect on phenotypes, which may explain the escape of dosage compensation in some genes. In addition, the enrichment of female-biased genes and the absence of male-biased genes on the X chromosome may also contribute to decreased X-chromosome expression in males ( fig. 6). Moreover, we also observed significantly higher levels of autosomal expression in males compared with females ( fig. 4B; supplementary table S10, Supplementary Material online; Wilcoxon P-values: head, 0.000017; antenna, 0.18; leg, 0.0061; gonad, 0.02). Therefore, higher autosomal expression may also lead to unbalanced expression of autosomes and X chromosome in males.
For gonad tissue, gene expression levels on the X chromosome were significantly lower in male testes than in female ovaries, reflecting the absence of dosage compensation in male testes. A lack of dosage compensation also has been found in Drosophila melanogaster testes, suggesting most of the dosage compensation complex genes are not expressed in the Drosophila germline; different testis cells also have different degrees of dosage compensation (Witt et al. 2021). Details of dosage compensation in testis cells and the associated molecular mechanisms require further analysis in L. striatellus. Thus, our results show that the degree of dosage compensation differs between somatic and gonad tissues of L. striatellus.
Two modes of dosage compensation have been observed in many other species, by downregulating gene expression of two X/Z copies in homogametic sex or upregulating gene expression of one X/Z copy in hemigametic sex. Downregulation of two copies of X results in unequal gene expression between autosomes and X chromosomes in homogametic sex; however, this phenomenon was not observed in female L. striatellus ( fig. 4A). Therefore, the dosage compensation mechanism of L. striatellus may be the upregulation of the male X chromosome.
In many species, sex-biased genes often exhibit a nonrandom genomic distribution. We found that the autosomes were rich in male-biased genes, while X chromosomes were rich in female-biased genes ( fig. 6A and B). Similar patterns have been found in Drosophila genus, and three possible mechanisms have been proposed to explain these patterns, including dosage compensation, sexual antagonism, and meiotic sex chromosome inactivation (MSCI). Lack of dosage compensation in the Drosophila germline is thought to be an important cause of male-biased gene defects in the X chromosome (Bachtrog et al. 2010;Meiklejohn and Presgraves 2012). For L. striatellus, a similar scenario is plausible, where the lack of dosage compensation in male testis results in an enrichment of female-biased genes on the X chromosome. However, this mechanism alone does not explain the observations in somatic tissues where female-biased genes were enriched on the X chromosome ( fig. 6A). In this context, it is possible that another evolutionary force sexual antagonism is at work, namely the opposing selection pressure between females and males may explain the deviations of gene distribution in somatic tissues. In the sexual antagonism theory, dominant mutations with beneficial fitness for females and detrimental fitness for males will be more likely to be fixed on the X chromosome in female homogametic organisms (Rice 1984). MSCI refers to the inactivation of X-linked genes during male meiosis. Since the X chromosome is inactivated in spermatocytes, genes required for sperm maturation are expected to escape to autosomes (Lu and Wu 2011). We observed significantly higher levels of autosomal gene expression in testis than in ovary ( fig. 4B), and that testisbiased genes were enriched in autosomes ( fig. 6B). Thus, genes critical for testis development appear to escape to the autosomes, which may indicate the presence of MSCI in SBPH testis; however, the existence of this process in testis remains to be verified.

Conclusion
This study provided an improved chromosome-level genome assembly of L. striatellus. We also confirmed that the degree of dosage compensation varied by tissues in L. striatellus. The complete dosage compensation regulation was present in somatic tissues but not in testis tissue. We also characterized the X-chromosome feminization of L. striatellus. Lack of dosage compensation in testis, sexual antagonism, and MSCI may contribute to the enrichment of female-biased genes on the X chromosome and an excess of male-biased genes on autosomes.

Insect Strain and Sample Preparation
The L. striatellus strain used in this study was initially collected from rice fields (in Hangzhou, China) in 2018 and reared in the laboratory for >10 generations. The strain was kept at 27 ± 0.5 °C with relative humidity >80% on rice seedlings (Xiushui 134) under a 16-h photoperiod.

Genome Sequencing
We used the long reads generated by Oxford Nanopore platform to do de novo genome assembly of L. striatellus. Genomic DNA from ∼500 male adult was extracted by QIAamp DNA MiniKit (Qiagen) following the manufacturer's instructions. The ONT sequence library was constructed by the ONT 1D Ligation Sequencing Kit (LSK109) according to the manufacturer's procedure and then sequenced on Oxford Nanopore PromethION sequencers. To correct the sequencing error of ONT data, and estimate the genome size, we further sequenced the DNA genomic extracted from 100 unmated male adults on Illumina platform HiSeq X. The Illumina sequencing library was constructed by the NEBNext Ultra II DNA Library Prep Kit.

Draft Genome Assembly
A genome survey was carried out to estimate the genome size and heterozygosity using the males Illumina paired-end reads by GenomeScope v2.0 (Ranallo-Benavidez et al. 2020), and the kmer was set to 19. The original draft genome was assembled by NextDenovo v.2.4.0 (https://github. com/Nextomics/NextDenovo), the NextCorrect module performed error correction, and the NextGraph module assembled the genome based on the ONT data, and parameters in configuration file were "rerun = 3; read_type = ont; read_cutoff = 1k; genome_size = 500m." The initial draft genome assembly was further polished twice to correct base errors. The first polish applied the raw Nanopore reads by NextPolish v1.3.1 with default parameters (Hu et al. 2020), and the second polish used Illumina reads by Pilon v1.2.3 with default parameters (Walker et al. 2014). The heterozygosity was reduced by HaploMerger2 with default parameters (Huang et al. 2017) and Purge Haplotigs v1.1.1 was further used to obtain the haploid contigs with parameters "-a 60" (Roach et al. 2018).

Hi-C Sequencing
Thirty male-fifth instar nymphs were collected for Hi-C library construction. To construct the Hi-C library, the samples were first mechanically disrupted with a homogenizer. The cross-linking reaction was conducted by incubating the cells in 2% formaldehyde. The reaction was stopped by adding 2.5 M glycine. Thereafter, the cells were flash-frozen in liquid nitrogen and stored at −80°C. After cross-linking, the chromatin was digested with 400 IU of MboI restriction enzyme (NEB). The Hi-C library was then prepared by adding biotin, adding T4 DBA ligase (NEB) to ligate DNA, adding proteinase K for reverse cross-linking, DNA fragments purification, fragment shearing to 300-500 bp, and DNA ends repair. Finally, DNA fragments labeled by biotin were separated on Dynabeads® M-280 Streptavidin. The Hi-C library was sequenced on an Illumina Hiseq platform. The Hi-C library construction and sequencing were performed by Novogene Co., Ltd.

Hi-C Assembly
Chromosomal-level assembly was constructed based on the 3D de novo assembly (3D-DNA) pipeline (Dudchenko et al. 2017). The Hi-C sequencing data were first aligned to the draft genome by Juicer version 1.6 (https://github. com/aidenlab/juicer) to obtain linkage matrix using "-s MboI." Then 3D-DNA version 180922 (https://github. com/aidenlab/3d-dna) was performed to obtain the chromosomal-level genome using "-r 3." Juicebox Assembly Tools (JBAT) v1.11.08 (Durand et al. 2018) was used to visualize and modify potential assembly errors before obtaining final high-quality genome. BUSCO version 5.1.3 (Simão et al. 2015) was used to evaluate the completeness of the genome assembly. Basic genomic features including GC content, gene density, and transcriptome reads coverage of ovary and testis were displayed by circos v0.69-8 (Krzywinski et al. 2009).

X-Chromosome Determination
In the L. striatellus sex chromosome determination system, females have two X chromosomes (XX), while males have only one X chromosome (XO). The X chromosome can be identified by calculating the coverage ratio between males and females. DNA sequencing coverage on the X chromosome in males was expected to be approximately half that in females at the same sequencing depth. Therefore, ∼100 unmated males and females were prepared separately for coverage analysis. Genomic DNA extraction and library construction were performed as described above and sequenced on the Illumina Hiseq X platform. Approximately 34G of males and 33G of females clean DNA sequencing reads were generated. The sequencing data were aligned GBE to the genome assembly by BWA v0.7.17-r1188 (Li 2013) with default parameters. The low-quality mapped reads were filtered out by SAMtools v1.10 using "-q 30" (Li et al. 2009). Read coverage was then calculated by sliding a 100-kb window across the genome using the command coverageBed from BEDTools version 2.25.0 (Quinlan and Hall 2010).

Transcriptome Sequencing
To better annotate the genome, we prepared the transcriptome of L. striatellus from representative tissues and developmental stages, and generated both long Nanopore and short Illumina RNA-seq reads. To construct ONT full-length cDNAs, RNA samples were prepared from different developmental stages of L. striatellus, including eggs, nymphs, adult females, and adult males. Samples from different body tissues, including carcass, fat body, gut, salivary gland, ovary, and testis, were prepared to sequence on the Illumina platform. All total RNA was extracted by the Takara RNIzol Total RNA Isolation Kit according to the manufacturer's protocols. The ONT and Illumina RNA libraries construction and sequencing were performed by Novogene Co., Ltd following the manufacture's instruction.

Dosage Compensation
Illumina RNA-seq data for gonad tissues (testis and ovary) were generated as described above, and two biological replicates were prepared for each. RNA-seq data for the somatic tissues leg, antenna, and head were downloaded from publicly available database with no biological replicate (Li et al. 2021). RSEM v1.3.1 (Li and Dewey 2011) was applied to calculate the FPKM expression matrix using default settings, and TMM normalization was applied across all samples using the run_TMM_scale_matrix.pl script in Trinity v2.11.0 (Haas et al. 2013). The relationship between biological replicates of gonad tissues was assessed by Spearman's rank correlation, with correlation coefficients of ∼0.97 for both ovary and testis (supplementary table S13, Supplementary Material online). The average expression of all replicates for each gene was used for the following analysis. We filtered genes with an average expression level of FPKM < 1 in males and females, as this approach has been used in dosage compensation studies for other species (Pal and Vicoso 2015;Catalán et al. 2018). We then log-transformed autosomal and X-chromosomal expression to obtain log 2 FPKM values in males and females. The Wilcoxon rank-sum test was applied to detect bias in female and male expression. The X:A expression ratio was calculated by dividing the median gene expression on the X chromosome by the median of all genes expressed on autosomes. The deviation of the female:male expression


Effect of the ACTN-3 gene polymorphism on functional fitness and executive function of elderly

During aging, physical integrity and cognitive abilities, especially executive function, become compromised, directly influencing the quality of life of the elderly. One good strategy to ensure healthy aging is the practice of physical exercise. Activities to improve aerobic capacity and muscle strength are extremely important in old age. However, some genetic factors can interfere both positively and negatively with these gains. In this context, the polymorphism rs1815739 (R577X) of the α-actinin 3 gene (ACTN-3) is commonly studied and related to muscle phenotype. Thus, the present study aimed to investigate the effect of the ACTN-3 gene polymorphism on the functional fitness (measured by the Senior Fit test) and cognitive capacity (evaluated by the Stroop test) of the elderly (n = 347), both men and women. We did not find the effect of genotype on functional fitness, but we did observed a positive effect of the ACTN-3 gene polymorphism on executive function. The presence of the X allele of the ACTN3 gene in the elderly was related to a better performance in the Stroop test (shorter answer time). Our results showed that ACTN-3 gene polymorphism affects the executive function of the elderly but not their functional fitness.

Introduction
Population aging is increasing worldwide. It is estimated that by 2050, the prevalence of individuals over 60 years of age will be twice as high as it is today. As a result, elderly individuals will represent about 2 billion of the world population (He et al., 2016). However, longevity is not always associated with a good quality of life. In fact, several comorbidities emerge from the aging process that negatively impact aged individuals, as .
/fnagi. . their functional abilities degrade (Blazer et al., 2015). Central nervous system damage, for example, accompanied by cognitive deficits is very common in the aged population (Erickson et al., 2019). Even individuals who are not affected by pathological conditions (e.g., dementias) experience cognitive decline as a result of aging (Harada et al., 2013). Cognitive decline begins with small cellular damages that in the long term extend to structural changes, such as gray matter atrophy and loss of synaptic density between brain areas (Oliveira et al., 2018;Erickson et al., 2019). In the prefrontal cortex, for example, it is possible to observe the loss of cellular bodies in early adulthood (Fjell et al., 2017). Damages to the prefrontal cortex result in cognitive deficits mainly related to executive functions (EF) (Zanto and Gazzaley, 2019). EF refer to the abilities to successfully perform independent, appropriate behavior, with a purpose of interest (Diamond, 2013). This includes a wide range of cognitive skills such as inhibitory control, selective attention, working memory, and cognitive flexibility (Diamond, 2013).
Changes in EF can be assessed in different ways. The Stroop test, for example, is a widely used tool to measure the central aspects of EF and to predict age-related cognitive decline (Fjell et al., 2017;Scarpina and Tagini, 2017). This test easily assesses the ability to inhibit interference, selective attention, and cognitive flexibility, related to frontal lobe dysfunctions that occur when processing a response to one stimulus simultaneously affects the processing of another response to the same stimulus (Fjell et al., 2017;Scarpina and Tagini, 2017).
Although cognitive functions become compromised during aging, there is robust evidence that the brain is capable of maintaining or recovering some functionality. One of the most commonly used tools to ensure cognitive integrity is physical activity (Blondell et al., 2014;Schlosser Covell et al., 2015;Veronese et al., 2016). Physical activity practice is a low-cost intervention, very effective in controlling age-related complications (Rebelo-Marques et al., 2018). The preservation of cognitive function from physical activity occurs through several molecular and cellular changes (e.g., increased levels of neurotrophic factors, decreased neuroinflammation, induction of neurogenesis, etc.), which influence neural systems involved in cellular signaling, information processing, memory retention, and learning (Loprinzi and Kane, 2015;Gomes da Silva et al., 2016;Henrique et al., 2018). It is important to emphasize that the protective effect of physical activity on the human brain occurs even when the practice starts after the brain maturation phase Prakash et al., 2015;Tolppanen et al., 2015). Indicating that the brain remains plastic enough to receive the benefits of an active lifestyle.
Besides cognitive protection, engaging in physical practice also ensures a better physical condition for aged individuals. During the aging process, the variables of functional fitness (strength, endurance, agility, and flexibility) tend to decrease naturally, compromising the independence of the individual in carrying out daily tasks (Legrand et al., 2014;Rebelo-Marques et al., 2018). For individuals aged 60 years or more it is highly recommended the practice muscle strengthening activities. This is because good muscle condition prevents sarcopenia and ensures better functional fitness-an individual's ability to perform activities of daily living (ADLs) safely and independently without the early onset of fatigue (Miljkovic et al., 2015;Erickson et al., 2019). Interestingly, muscle strength is also associated with cognitive response in the elderly. For instance, high levels of isokinetic strength are related to better performance of general cognitive function assessed by the Mini-Mental State Examination (MMSE) (Nakamoto et al., 2012). Upper and lower limb strength is also associated with higher scores on cognitive test batteries (Steves et al., 2016), while low levels of muscle mass and strength are associated with poor cognitive performance (Herold et al., 2019).
Genetic factors influence muscle characteristics. For instance, the ACTN-3 R577X polymorphism is commonly related to muscle phenotype. The ACTN3 gene is responsible for encoding the α-actinin-3 protein (Zempo et al., 2010;Pickering and Kiely, 2018). Actinin-3 is a structural protein, a component of the Z disk of muscle fiber, which acts to anchor actin filaments within the sarcomere (Yang et al., 2011). Its expression is restricted to skeletal muscle (type II, fast glycolytic fibers) and, as a consequence, individuals with a deficiency in this protein tend to have a lower percentage of these fibers (Vincent et al., 2007). The R577X SNP results in a C to T base substitution, which leads to the alteration of an arginine base (R) to a premature stop codon (X) (North and Beggs, 1996;North et al., 1999;Yang et al., 2003;Pickering and Kiely, 2018). An estimated 20-30% of the world population (over 1 billion people worldwide), are deficient in α-actinin-3 (North et al., 1999;Mills et al., 2001;MacArthur et al., 2007). There are three possible genotypes: RR and RX-which seem to confer muscle strength predisposition to the individual and XX-which is the total absence of the protein and it is more related to decreased strength and loss of muscle function (Mills et al., 2001;Clarkson et al., 2005;Pickering and Kiely, 2018). These muscle phenotype differences are observed in elite athletes, where individuals who are homozygous for the X allele are underrepresented in speed, power, and strength modalities (Papadimitriou et al., 2019).
Taking into account the important role of muscle strength in healthy aging, we aimed to investigate the effect of the R577X polymorphism of the ACTN-3 gene on functional fitness and executive function in aged individuals. Functional fitness was assessed using the Senior fit test protocol. For the executive function response, the Stroop test was used. Other variables such as years of educational level, anthropometric measurements, and health information were also collected for analysis. Understanding the factors that compromise executive functions in old age can help us to create strategies that can be adopted to increase the functional independence and quality of life of the elderly.

Materials and methods
We conducted a cross-sectional study of convenience and non-probabilistic sampling approved by the ethics committee of the Federal University of São Paulo (CEP/UNIFESP -CAAE: 0630/2018). To be included in this study, the person had to sign an Informed Consent Form and a medical authorization (issued for up to 1 year) to perform physical tests (requirement of elderly centers). As for exclusion criteria, those who had musculoskeletal and/or neurological diseases that made it impossible to perform the functional capacity tests or were being treated for cognitive, psychological, or psychiatric illnesses and alcoholism were excluded. Therefore, 347 individuals, aged 60 years or more, of both sexes were recruited from an elderly center in the Alto Tiete region of São Paulo state.

Questionaries and anthropometric data
It was asked through questionaries, information regarding educational level, 12-month fall recall, and general health was provided. Body weight and height were collected to determine body mass index (BMI) by the following equation:

General functional fitness index
The functional fitness of the volunteers was evaluated by the Senior Fit test method, which is composed of a battery of tests that includes measures of strength, aerobic resistance, flexibility, agility, and dynamic balance. The score is established according to the criteria of each test. Each test has a rating of five categories, according to the participant's performance, gender, and age. In the end, GFFI-an indicator calculated through the sum of the points obtained in each test-is generated. The general classification is ranked from worst to best, receiving the following classifications: very weak, weak, regular, good, and very good.
For our analyses, the categories very weak and weak, and good and very good were grouped to increase the representation of participants in these categories. Thus, the final classification adopted for the IGAF as well as for the individual tests had three levels, namely, very weak/weak, fair, and good/very good.
The tests were applied in the following order: (1) sitting to standing, (2) elbow flexion, both for muscle strength evaluation, (3) time up and go (TUG), for analysis of sitting, balance, position transfers, stability, and change of gait course, and (4) a 6-min walk (for aerobic capacity).

Stroop test
The adapted version of the Stroop test was used as a tool to assess the executive function of the volunteers. The validated model by Kulaif and Valle (2008) with numbers and colors, adapted from the Victoria version (Tremblay et al., 2016), is very useful in the presence of unlettered participants. Prior to the test's beginning, it was assured that the participant was able to recognize/distinguish the colors and numbers of the test without hesitation, to validate the results. The test consists of 4 cards. In the first (card 1-colors), the participant is asked to quickly name the colors rectangles (red, green, blue, and black). Then, in the second card (card 2-colors and numbers) numbers were added to the colored rectangles like in card 1, at this moment the participant is asked to name the respective number or the color of the rectangles. The third card (card 3-Stroop Effect) consists of colored numbers (same colors as card 1), the participant is then asked to name the colors of the number or the respective number when they were black. At this stage, a certain degree of interference may be unavoidable. Thus, to further minimize any frustration, the last card (card 4) with only numbers colored in black, that should be named was added to our model. The evaluation process considers the time spent on each card, as well as the number of errors. When the error or spontaneous self-corrected, we considered it right.

ACTN-polymorphism analysis
Epithelial cells from oral mucosal were collected by mouth scraping (buccal swab). DNA extraction was performed following the chelex resin protocol for mouth swabs (Ostheim et al., 2020;Yan et al., 2022). Quantification was performed on a NanoDrop R spectrophotometer -ND1000. The DNA used in the standard amplification reactions was diluted in autoclaved ultrapure water. The analyzes of the selected genotypes were performed by the allelic discrimination method in the real-time-PCR (Polymerase Chain Reaction) equipment, using Taqman R SNP genotyping assays (Applied Biosystems, Foster City, CA).

Statistical analysis
To characterize the sample, education, nutritional status, functional fitness, and respective GFFI, the general linear model (GLM) or Chi-square test was used. For GLM, the results are presented as the mean and standard deviation of the mean followed by the F value. The Chi-square test was conditioned on the interpretation of the residual (R = observed value minus the expected value) and the adjusted residual (RA), when significant, followed by the X² value. To analyze the . /fnagi. . effect of genotype on muscle strength and aerobic capacity, ANCOVA analysis was used, with age as a covariant. As for executive function and the effect of functional fitness on the response to the Stroop test, a linear regression was performed with the following independent variables: age, schooling, sex, genotype and the tests: elbow flexion, sitting and standing, and walking for 6 min. For the elbow flexion and sit and stand tests, the number of repetitions was analyzed, while for the 6-min walk, we used the distance in meters. The analysis of the models was performed through the interpretation of the standardized and adjusted coefficient (β), followed by the analysis of the t value and significance value. When the independent variables presented more than one possibility of classification, we fixed one of them. For all statistical tests, it was adopted a level of significance lower than 5% (p ≤ 0.05). Table 1 presents the descriptive data of the sample. The genotype distribution of the ACTN3 R557X polymorphisms was 110 (31%) for the RR genotype, 148 (42%) for the RX genotype, and 89 (25%) for the XX. The mean age between the groups was similar, with no statistically significant difference. Nutritional variables and schooling also showed no significant difference between the genotypes studied.

Results
The functional fitness of the participants was assessed using the Senior Fit test. In our analysis, we used the elbow flexion, sit and stand, TUG, and 6-min walk tests. To increase the representation of participants in the categories, some classifications were grouped. Thus, the classification adopted in this study had three categories: very weak/weak, regular, and good/very good. In Table 2, we see the results of functional fitness. There was no significant difference between the groups. It is possible to see that the majority of the elderly were classified as regular in all genotypes. Indicating that there is no interference of the genotype in the classification of functional fitness. When verifying the number of repetitions in the lower and upper limb strength tests and the walked distance (meters) in the aerobic test, we also did not observe a significant effect of the genotype (Table 3). The prevalence of falls in the elderly is also an indication of the individual's functional quality. Thus, we collected information on 12month fall recall, but there was no genotype effect on this variable.
Executive function was assessed through the Stroop test. We performed a linear regression to verify the factors that influence the volunteers' executive functions (Table 4). We can observe that age and education are factors that influence the answer's time in card 3. As the person ages, the response time for card 3 increases (p = 0.003). Regarding education, the higher the educational level, the better the response in the Stroop test (p < 0.001). As for the genotype, RR individuals have a worse cognitive performance (they answer the test for a longer time) when compared with RX and XX individuals (p = 0.05 for both) (Tables 4, 5). This result suggests that the presence of the X allele of the ACTN3 gene is related to a better performance of the executive function.  Data expressed by percentage (%). X², Chi-square; GFFI, General functional fitness index.

Discussion
The present study aimed to investigate the effect of the R577X polymorphism of the ACTN-3 gene on functional fitness and executive function in the elderly. For this, we recruited 347 volunteers, both sexes, aged 60 years old and over. The R577X polymorphism results in the change of the R allele for a premature stop codon X, which leads to protein deficiency, thus influencing muscle phenotype (Pickering and Kiely, 2018). In our studied population, we did not observe any differences between functional fitness tests and the different genotypes of the ACTN-3 gene. Even though the presence of the X allele is associated with decreased physical performance, increased frailty (Ma et al., 2018), loss of muscle function  Data expressed as mean and standard deviation (M ± SD). (Kikuchi et al., 2015) and an increased risk of sarcopenia (Cho et al., 2017), our volunteers showed similar performance regardless of individual genotype-both in muscle strength tests (elbow flexion, sit and stand) and in aerobic capacity tests (6-min walk). Similarly, a cohort study that evaluated the association between the ACTN-3 gene and different muscle phenotypes in more than a thousand volunteers found no influence of the ACTN-3 genotype on muscle phenotypes (Delmonico et al., 2008). However, when evaluated over 5 years, a decline in muscle function with a significant genotypic association could be seen. Individuals with the XX genotype showed a greater decline in muscle function (Delmonico et al., 2008). As the volunteers in our sample belong to an elderly community center where they participate in various sports .
/fnagi. . activities, regular training may have conferred benefits on the physical integrity of the elderly, regardless of their genetic predispositions. As found in the study by Moraes et al. (2018), differences in muscle strength were not found between the genotypes of the ACTN-3 gene, however, after a physical exercise intervention, there was an improvement in muscle function even in individuals with protein deficiency (Moraes et al., 2018). These findings indicate that the regular practice of physical activity in the elderly population is capable of attenuating possible muscle losses related to the ACTN-3 gene polymorphism. Reinforcing the importance of good maintenance of muscle function for the elderly.
In addition, muscle strength has been studied as a predictor of cognitive performance in aging. In a recent study carried out by our group, we observed that functional fitness, in addition to age and education, was one of the predictors of the cognitive performance of the elderly, assessed by the Stroop test (Braga et al., 2022). Among fitness tests, muscle strength appears as a potential predictor of a better cognitive response (Braga et al., 2022). The mechanism by which muscle strength influences cognition is still not well-understood. However, several studies show a relationship between these two variables. In a Brazilian study, 62 participants underwent 24 weeks of strength exercise and, after the intervention, tests of executive function as well as short-and longterm memory were applied. It was observed that volunteers who participated in the intervention showed improvement in neuropsychological tests (Cassilhas et al., 2007). The same association was found in the Koreans elderlies. This longitudinal study, which followed participants for 6 years, suggests that poor handgrip strength is significantly associated with an increased risk of cognitive dysfunction in aging (Jeong and Kim, 2018). However, none of these studies evaluated a genetic component along with physical capabilities in the cognitive response during aging.
Based on this, we sought to investigate whether the ACTN-3 gene can be a predictive factor for the executive response in the elderly. As individuals with the RR genotype have characteristics of greater muscle strength, we hypothesized these individuals would perform better in executive function. Interestingly, participants with the RR genotype did not have a good executive performance, answering the test in a longer time when compared to individuals who had the ACTN-3 (XX) gene polymorphism. As one of the characteristics of the XX genotype is a better cardiorespiratory performance, it may indicate that these individuals are more likely to present positive cognitive results, since a good aerobic capacity contributes to healthy brain aging (Hayes et al., 2013). The prevalence of the XX genotype is increased in the population as a result of the evolutionary adaptations to cold climate regions (Wyckelsma et al., 2021). Therefore, this could be a speculative suggestion that the effect on cognitive performance could have been derived from a positive selection of the X allele. Also, as the glycolytic fibers present accentuated degradation with aging (Milikovic et al., 2015), the effects of the RR genotype on cognition in older individuals may not be so apparent. These results are interesting and shed light on further investigation, for instance, it would be interesting to investigate these relationships in different domains of cognition. Due to the number of participants and research volunteers that worked in our study, we were unable to perform further cognitive analyses. For this reason, we limited our evaluation to the executive function. Also, it would be interesting to investigate other genetic factors that may be involved in muscle characteristics. The ACTN-2 gene, for example, in some cases, can partially supply the deficiency of the α-actinin-3 protein (MacArthur et al., 2007), and in this case, may be responsible for maintaining physical function. The knowledge of genetic markers or factors that can influence the cognition and functional fitness of the elderly, can be helpful for the creation of personalized intervention strategies to suit the individual needs of this population.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

Ethics statement
The studies involving human participants were reviewed and approved by Comitê de Ética em Pesquisa -UNIFESP (CEP/UNIFESP -n:0630/2018). The patients/participants provided their written informed consent to participate in this study.

Author contributions
JS participated in the study design, data collection and analysis, interpretation of results, discussion, drafting, and manuscript writing. PG, SS, and RA participated in the study design, data collection, and intellectual contribution. NS and ID participated in the data analysis. CM participated in data analysis, interpretation of the results, and critical review. SG participated in the supervision of the study, data interpretation, and critical review. All authors contributed to the article and approved the submitted version.