The mammalian NDR kinases are known regulators of the cell cycle, which implicates them directly in a key aspect of cellular senescence. In vitro, both NDR1 and NDR2 interact with the CyclinD1/CDK4 complex, which drives cell cycle progression. Mechanistically, CyclinD1 has been shown to increase the kinase activity of NDR1/2 by reducing its autoinhibition and promoting the G₁/S cell cycle progression (Du et al., 2013). It is known that in the canonical Hippo pathway, NDR1/2 is activated by phosphorylation in the Thr444/Thr442-residue by the MST1/2 kinases. Moreover, in vitro work shows that NDR1/2 can also become phosphorylated by MST3 exclusively in the G₁ phase. This phosphorylation controls the NDR1/2 kinase activity on p21 during the cell cycle, as it was demonstrated that depletion of NDR1/2 increased p21 stability and induced G₁ arrest (Cornils et al., 2011).

The other mammalian NDR kinases, LATS1 and LATS2, have also been found to participate in the cell cycle transition. It was shown that, independently of its kinase activity, LATS1/2 can inhibit human cancer cell proliferation and induce a G₂/M arrest. The mechanism by which this occurs is yet not completely understood, but research suggests that LATS1 and LATS2 act independently in distinct pathways in cell cycle progression. LATS1 binds to Cell division control protein 2 (CDC2) and inhibits the kinase activity of both Cyclin A/CDK1 and Cyclin B/CDK1 protein complexes. On the other hand, LATS2 decreases the kinase activity of the Cyclin B/CDK1 and the Cyclin E/CDK2 complex exclusively. The consequence is that overexpression of LATS1/2 can prevent the G₂/M transition (Yang et al., 2001) and act as a negative regulator of the cell cycle progression. A finding that could seem contradictory was later revealed in a model of replicative senescence of human fibroblasts, which shows an increase in AMP-activated protein kinase 5 activity, and the subsequent phosphorylation of LATS1 at the S464, decreases LATS1 stability. Accordingly, inhibition of LATS1 activity accelerated replicative senescence (Humbert et al., 2010). This suggests that there are several pathways in which LATS1 might exert their role in the regulation of the cell cycle. A strong indication of this comes from the fact that overexpression of AMPK5 did not affect p53 activity, thus it is likely that the p53-p21 axis is not involved in this pathway. Arguably p16, a selective inhibitor of CDK4 and CDK6 kinases, is the most well-characterized CDK inhibitor that participates in the cell cycle arrest of senescent cells, mainly in the G₁/S transition. This is achieved by maintaining the CDK4/6 downstream target, the retinoblastoma protein (pRB) in a hypophosphorylated state (Safwan-Zaiter et al., 2022).

Even in a mouse neuronal cell line, p16 expression has been associated with resistance to apoptosis when cell cycle regulators like Cyclin D1 are overexpressed in terminally differentiated cells (Kranenburg et al., 1996). Besides the inactivation of the pRB, it has been shown that in human fibroblasts, p16 activation leads to an increase of reactive oxygen species (ROS) mediated by a positive feedback loop that involves the downstream activation of the protein kinase C delta and downregulation of LATS1, in a mechanism that irreversibly blocks the cell cycle progression (Takahashi et al., 2006). In this same direction, it was reported that LATS2 participates in the repression of E2F genes in cellular senescence after expression of pRB, particularly from the dimerization partner, RB-like, E2F and multi-vulval class B (DREAM)-repressor complex, and the formation of the senescence-associated histone foci (SAHF) and the increased beta-galactosidase activity, characteristic of senescent cells. Remarkably, it was also found that both RB1 and LATS1 loci are mapped in close proximity to the 13q locus, which is often lost in tumourigenic cell lines and can impair the induction of cell cycle arrest (Tschöp et al., 2011). Finally, another key piece of evidence that links NDR kinases in the permanent arrest of the cell cycle in senescent cells comes from a series of papers published by Aylon et al., in which they use multiple murine and human cell lines to investigate and describe a pathway that involves the translocation of LATS2 from the centrosomes to the nucleus in the context of mitotic stress, where it binds to Mouse double minute 2 homolog (MDM2), thus increasing p53 stability and transcription of downstream targets such as CDKN1A (p21) and LATS2, creating a positive feedback loop that helps to maintain the cell cycle arrest (Aylon et al., 2006). Moreover, they showed that LATS2 deficiency allows cells to escape oncogene-induced senescence (OIS) by HRAS (Aylon et al., 2009). The malignant transformation of cells is often linked to intrinsic causes, such as mutations in the DNA, and among them, mutations in RAS genes are one of the most aggressive and main risk factors of cancer. Interestingly, the expression of KRAS is sufficient to increase the NDR1 protein levels, and inhibition of NDR1 induces apoptosis and also increases p21 and LATS2 levels, possibly as a compensatory mechanism.

These data together suggest that although NDR kinases might have distinct roles within the pathways, there is a wide overlap of functions. It has also been shown that NDR2 mRNA levels are increased in pancreatic cancer patients which allows for malignancy of transformed cells (Grant et al., 2017). Although this observation is contradictory to the role of NDR kinases in inducing cell cycle arrest in cellular senescence, it suggests that in cells that escape the cell cycle arrest, NDR kinases might allow for survival and an increase in the malignity of the tumors by hyperactivation of growth and facilitating migration possibly through MOB-mediated pathways and Hippo signaling. For example, it was shown that human MOB2 competes with MOB1 for binding to NDR1/2 as a negative regulator (Kohler et al., 2010) in a model of DNA damage and that independently of NDR1/2, MOB2 promotes survival and the cell cycle progression in human cells through inhibition of p21 and p53 (Gomez et al., 2015). Supporting this idea, it was shown that, unlike MOB1 or MOB2, MOB3 inhibits LATS1/2 signaling within the Hippo pathway and allows for continued proliferation of cells even after OIS (Dutchak et al., 2022), potentially contributing to cancer progression. This is consistent with the observation that LATS2 enforces cell cycle arrest during OIS by engaging with pRB and promoting the silencing of E2F genes (Tschöp et al., 2011). In summary, it is evident that NDR kinases play a significant role in cell cycle regulation through various mechanisms across different species. While their direct involvement in the permanent cell cycle regulation of cellular senescence has not been directly addressed, we suggest that they could be crucial components of the machinery enforcing the cell cycle arrest in senescent cells.

Many studies have examined the complementary and yet contradictory relationship between cellular senescence and apoptosis mechanism during aging. The current consensus in the field is that both mechanisms have evolved as a regulatory process for tissue modeling and to prevent the malignant transformation of cells (Campisi, 2013), thus many of the molecular mechanisms that induce apoptosis, cellular senescence or apoptosis resistance in senescent cells overlap and are governed by the same proteins such as p53, BCL-2 or extracellular-signal-regulated kinases (ERK) signaling (Childs et al., 2014). It has been hypothesized that one main factor that determines whether the cell will enter senescence or apoptosis relates to cellular stress levels. Extreme cellular stress resulting from various factors such as irreversible DNA damage, exposure to UV irradiation, oncogenic activation, or withdrawal of growth factors triggers the intrinsic apoptotic pathway (Visser and Yang, 2010). It has been shown that both human LATS1 and LATS2 participate in parallel but independent apoptosis mechanisms, one in which LATS1 engages p53 independently of BAX which increases caspase 3 activity (Yang et al., 2001), and the other in which LATS2 overexpression downregulates BCL-2 and BCL-xL, resulting in a cascade that increases caspase 9 processing (Ke et al., 2004).

Other additional pathways in which LATS1/2 has been shown to be involved in the intrinsic apoptotic pathway are the upregulation of LATS2 by Checkpoint kinase 1 (CHEK1) upon activation of the DNA Damage Response (DDR) (Aylon et al., 2009), and the activation of the protease OMI/HTRA2 by LATS1, which is required for caspase cascade activation upon mitochondrial permeabilization (Kuninaka et al., 2005). Except for the case of excitotoxicity, aging neurons are highly resistant to stress and apoptosis (Kole et al., 2013), probably as a consequence of some of them becoming senescent (Si et al., 2021). Activating Transcription Factor 4 (ATF4) plays an important role in neuronal apoptosis and it is known that it induces the transcription of the C/EBP homologous protein, which activates the protein p53 Upregulated Modulator of Apoptosis (PUMA) in mouse cortical neurons (Galehdar et al., 2010). Additionally, it was shown both in mouse and human cell lines that EI2F-α promotes the translation of ATF4 and subsequent stabilization of LATS1 which increases cell death after oxidative stress (Rajesh et al., 2016). A downregulation on this pathway has yet to be demonstrated as a possible link between NDR kinases and the resistance of apoptosis in aging neurons.

SASP is a senescence state characterized by the secretion of a diverse array of molecules, ranging from pro-inflammatory cytokines to growth factors. While research on neuronal SASP is limited, compelling in vitro evidence from rat primary neurons suggests that senescent neurons also undergo SASP, a process driven by the transcription factor GATA Binding Protein 4 (GATA4) and impaired autophagic flux (Moreno-Blas et al., 2019). The potential involvement of NDR kinases in the regulation and secretion of SASP remains largely unexplored. However, NDR kinases have been associated with the regulation of established SASP components, such as Tumor necrosis factor-alpha (TNF-α) which has been implicated in the heightened neuroinflammatory response and cognitive impairment during aging (Habbas et al., 2015; Probert, 2015). It has also been shown that murine NDR1 positively regulates the production of TNF-α and Interleukin 6 (IL-6) by directly binding to Smad ubiquitination regulatory factor 1 (SMURF 1) which leads to ubiquitination and degradation of mitogen-activated protein kinase 2 (MEKK2) and reducing the transcription of its downstream targets (Wen et al., 2015). Besides NDR2, another study reported that NDR1 activity also increases TNF-α dependent activation of the transcription factor nuclear factor “kappa-light-chain-enhancer” of activated B-cells (NF-κB) in various human tumor cell lines (Shi et al., 2012), supporting the important role of NDR1/2 in the regulation of TNF-α activity. Interestingly, Braitsch et al. (2019) found an opposing role for LATS1/2 in NF-κB regulation and reported that LATS1/2 KO increases NFκB activation and vanin1 expression, which favors epithelial-mesenchymal transition. NFκB is one of the main transcription factors that accumulate in the chromatin of senescent cells and controls the expression of SASP genes (Chien et al., 2011; Freund et al., 2011), suggesting an important link between NDR kinases and the control of the SASP.

In summary, NDR kinases serve as crucial regulators of the cell cycle. While not directly addressed, the existing literature strongly points out their involvement in enforcing cell cycle arrest in senescent cells. When this effect is circumvented, the expression of NDR kinases can have deleterious consequences, aiding cells in survival and migration, particularly through their role in the Hippo pathway. Additionally, it is plausible that NDR kinases participate in the pathways dictating whether a cell enters senescence or undergoes apoptosis and eventually providing senescent cells with resistance to apoptosis. A maladaptive feature of this role is that increased NDR expression is often observed in transformed cells due to its pro-survival effect, facilitating tumor progression and malignancy. Finally, it is well-known that mechanistically, NDR kinases can participate in the increased inflammatory activity during aging, and possibly in the control of SASP. As documented in the previous literature, functions of NDR1/2 and LATS1/2 have been associated with contradictory roles, which is compounded by a general lack of insight into their essential role as regulators of aging, a point that is highlighted throughout this review.

There is enough evidence in the literature that links NDR kinases to AMPK signaling, which is required for the proper function of AMPK signaling, one of the main pathways that cells have to respond to nutrient deprivation. Moreover, NDR activity within the pathway has also been shown to play a maladaptive role, particularly by promoting age-related diseases such as metabolic syndrome. Besides nutrient sensing, activation of the AMPK pathway with metformin prolongs lifespan across species (Novelle et al., 2016), counteracts the development of neurodegenerative diseases (Rotermund et al., 2018) and in middle-aged mice improves cognitive performance by increasing autophagy of the hippocampus (Kodali et al., 2021). Additionally, clinical studies suggest that metformin treatment inhibits memory loss in diabetic adults (Ng et al., 2014). In vitro, evidence shows that the activation of the AMPK pathway by nutrient starvation, metformin treatment, or its more potent analog phenformin increases the phosphorylation and degradation of the NDR kinase downstream target YAP, either by direct phosphorylation of YAP on its Ser-94 residue and thus disruption of its interactions with TEAD or indirectly through increase of LATS1/2 activity (DeRan et al., 2014; Mo et al., 2015). In this line, it was also reported that silencing of LATS1/2 is sufficient to ablate the effect of energy stressors on AMPK-dependent YAP phosphorylation (DeRan et al., 2014). Interestingly LATS1/2 is not always essential for the activation of YAP via AMPK signaling since it was shown that AMPK can inhibit the oncogenic transformation of LATS-null mouse embryonic fibroblasts by directly inhibiting YAP activity (Mo et al., 2015).

Similarly, in Drosophila melanogaster larval central brain and ventral nerve cord, it has been shown that AMPK together with the AMPK upstream regulator liver kinase B1 (Lkb1), can inactivate Yki independent of the NDR Kinases (Gailite et al., 2015). AMPK signaling also seems to play a role in the shuttling of human YAP. A signaling cascade that involves AMPK, LK1B and LATS1/2, promotes the interaction of YAP with the WNT signaling protein Disheveled, and its eventual nuclear export (Lee et al., 2018). The mechanism involves the activation of AMPK, the downstream increase of SCRIB (Liu et al., 2020), Angiomiotin-like protein 1 and 2 (AMOTL1/2) and Angiomotin (AMOT) (DeRan et al., 2014) protein levels, which are involved in LATS1/2 stabilization and therefore AMPK-dependent YAP degradation. Furthermore, another study showed that Klotho^± mice, a widely used knockout mouse model in aging research that displays extremely short life span and premature aging (Kuro-o et al., 1997) display a weaker AMPK-LATS1 interaction as well as decreased AMPK-dependent phosphorylation of YAP (Luo et al., 2023). Although the exact mechanism has not been described yet, these data suggest that there is a direct interaction between AMPK and LATS1 which might be facilitated by the anti-aging protein Klotho.

The other mammalian NDR kinases have also been linked to AMPK signaling. Overexpression of NDR1 in mice decreases AMPK and the downstream Acetyl-CoA-carboxylase phosphorylation, which leads to enhanced de novo lipogenesis and increased incidence of non-alcoholic fatty liver diseases (NAFLD). In accordance, NDR1 overexpression alone is sufficient to cause NAFLD and increase inflammation in the liver under a regular diet. Interestingly, NDR1 exhibits a deleterious effect on the disease. A liver-specific KO of Stk38 ameliorated high-fat-died (HFD) induced insulin resistance, hepatic inflammation, and lipid accumulation, as well as can reduce the cholesterol and triacylglycerol (TAG) levels (Rawat et al., 2023), which are major indicators of metabolic syndrome. Moreover, besides a systemic effect, NAFLD has been linked to reduced cognitive functions in adults (Bertolotti, 2014; Seo et al., 2016; Takahashi et al., 2017; Weinstein et al., 2018, 2019) and in mice, HFD has been shown to impair amygdala and hippocampus-dependent memory consolidation and cause neuroinflammation during aging (Spencer et al., 2017). Additionally changes in cholesterol as well as TAG levels have been linked to the neuronal aging process and the etiology of various age-related diseases including Alzheimer's (AD), Parkinson's (PD) and Huntington's disease (Spitler and Davies, 2020; Nunes et al., 2022). It has been extensively demonstrated that the responsiveness of rodent AMPK to nutrient deprivation decreases during aging (Salminen and Kaarniranta, 2012). Based on previous studies demonstrating the significant interactions of AMPK and NDR signaling, we suggest that the age-related reduction of AMPK signaling might involve a decreased downstream inhibition of YAP by NDR-kinases and increased lipid accumulation and higher incidence of NAFLD observed during aging.

The complex intercommunication between mTOR signaling and NDR kinases has been intensively studied both in Drosophila and in mammals. The first study that linked NDR-Kinases to mTOR showed that Drosophila salivary gland-specific KO of wts leads to decreased cell death, caspase activity and autophagy, while the expression of a dominant negative form of Drosophila Tor, abolished the effect of the wts KO (Dutta and Baehrecke, 2008). Furthermore, a substantial body of evidence indicates a two-way communication between NDR kinases and TOR signaling, with particular relevance for neuronal function.

In Drosophila Trc phosphorylation at T449 was shown to be dependent on TorC2 in class IV sensory neurons and required for the regulation of dendritic tilting (Koike-Kumagai et al., 2009). Along those lines, it is known that semaphorins promote neuronal substance adhesion in Drosophila by blocking dendrite crossing in a signaling cascade that involves the semaphorin receptor Sema-2B, TorC2, the β-integrin-subunit myospheroid (Mys), and Trc (Meltzer et al., 2016). Additionally, trc KO displays increased synaptic boutons number in the neuromuscular junction (NMJ), and a decrease in Yki phosphorylation therefore increasing Yki-dependent transcription of the Wiskott-Aldrich Syndrome Protein (Wasp) (Natarajan et al., 2015). It is well-known that Wasp regulates the synapse development in the neuromuscular juntion (Coyle et al., 2004; Khuong et al., 2010; Nahm et al., 2010)through actin polymerization (Stradal et al., 2004), hence a model was proposed where Trc acts downstream of TorC2 and regulates Wasp levels and modulates actin polymerization and synapse formation (Natarajan et al., 2015). In that regard, an accumulation of filamentous actin (F-actin) in the Drosophila brain has recently been suggested to occur during aging and disruption of actin polymerization in aged animals rescues autophagy levels, restores the youthful neuronal cell phenotype, and slows brain aging (Schmid et al., 2023). Besides their role as essential regulators of development in mammals (Kramer et al., 2022), the Drosophila Wasp homolog has been implicated in the increase of neuronal F-actin during aging in the brain, which suggests that their activity could be related to the pathological loss of proteostasis and deregulated nutrient sensing.

Another interesting role of NDR kinases in mTOR signaling is that NDR1 has been shown to increase mTOR-Complex 1 (mTORC1) activity and Rabin8 phosphorylation, leading to autophagy inhibition (Amagai et al., 2015), while on the other hand, LATS2 participates in the suppression of mTORC1 activity (Gan et al., 2020), which is implicated in pancreatic β-cell apoptosis and autophagic cell death under diabetic conditions. Additionally, the same study found that Lats2 KO rescues high fat-died induced phosphorylation of the ribosomal S6 protein and p62 accumulation, which stems from increased mTORC1 signaling and impaired autophagic flux, respectively. Besides mediating autophagy-dependent effects under diabetic conditions, LATS2 is also colocalized with autophagosomes and accumulates upon treatment with bafilomycin or chloroquine, which are commonly used inhibitors of lysosomal function, suggesting that LATS2 is degraded by the autophagosome-lysosomal machinery (Yuan et al., 2021). Although not fully characterized, these data suggest an evolutionary conserved regulatory axis of NDR kinases by Tor/mTOR that participates in neuronal function and disease.

The IIS is one of the major nutritional signaling pathways and has been thoroughly implicated in lifespan control, aging and age-related pathologies across several species (Altintas et al., 2016; Mathew et al., 2017; Zia et al., 2021). The canonical activation of IIS commences with the binding of either insulin or IGF1 to the insulin receptor, leading to autophosphorylation of its cytoplasmic tyrosine residues. Subsequently, adapter proteins such as Insulin-receptor-substrates (IRS) bind to the phosphorylated residues, initiating the activation of the Phosphoinositide 3-kinases (PI3K), Protein kinases B (PKB/AKT, hereafter referred to as AKT), and mTOR cascade which results in the downregulation of forkhead box O (FOXO) transcription factors. This constitutes one of the best-defined regulatory networks central to the control of lifespan and longevity (Wang et al., 2014; Webb and Brunet, 2014; Klotz et al., 2015; Martins et al., 2016). The IIS has been shown to play a neuroprotective role during mammalian brain aging, and low levels of IGF1 are linked to several age-related diseases (Zia et al., 2021).

Within this regulatory axis, the activation of AKT has been demonstrated to reduce MST1 and LATS1 activity. Similarly, inhibition of the PI3K-AKT pathway leads to an increase in MST1 and LATS1 phosphorylation that enforces cytosolic localization and degradation of YAP. Intriguingly, in a non-phosphorylated state, YAP remains in the nucleus and downregulates the phosphatase and tensin homolog (PTEN), a negative regulator of the PI3K-AKT pathway. This intricate interplay forms a positive feedback loop that illustrates the complex and nuanced regulation within this signaling network (Qian et al., 2021). AKT activation and its downstream effect on NDR-Kinases seem to be dependent on the DNA-double-strand-break-repairing protein DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), as suggested by experiments in human glioblastoma cell lines. Inhibition of DNA-PKcs decreases AKT phosphorylation at its S473 residue and NDR1 activation upon glucose deprivation. Additionally, AKT and DNA-PKcs-dependent activation of MST1 in these conditions increases phosphorylation of NDR1 at S281 and T282 (Shiga et al., 2020). Supporting the role of NDR kinases in enforcing a positive feedback loop on AKT signaling, adenoviral overexpression of NDR1 in mice was shown to reduce the activation of AKT by phosphorylation at S473 and T308. This resulted in impairment in glucose-dependent Insulin signaling and increased inflammation, demonstrated by higher levels of Interleukin-6 and TNF- α (Rawat et al., 2023).

Even though it has not been demonstrated yet, this could mean that an increase or impairment in the function of NDR kinases during aging might have a maladaptive role which promotes the age-related loss of cognitive function through an increase in ISS.

Sirtuins are nicotinamide adenine dinucleotide (NAD⁺) dependent protein deacetylases that have been thoroughly implicated in the regulation of aging and lifespan. It has been demonstrated in models ranging from C. elegans to humans that SIRT1 function and protein levels decrease with age and that overexpressing or reconstituting SIRT1 function can increase lifespan and delay aging (Satoh et al., 2013; Kilic et al., 2015). Moreover, the age-related alteration of autophagy has been linked to a decrease in SIRT1 (Xu et al., 2020), and the other way around, SIRT1 plays an essential role in the regulation of mammalian autophagy through the regulation of several key steps of the autophagic pathway (Kitada et al., 2016). In the brain, SIRT1 is predominantly expressed in neurons within the hippocampus and plays a crucial role in memory and plasticity (Michán et al., 2010). In human cell lines, it was shown that the acetyltransferases p300 and CREB-Binding Protein (CBP) can specifically acetylate NDR2 at K463, while SIRT1 is the major deacetylate of NDR2 (Tang and Yu, 2019). A similar study showed that LATS1 is also under the regulation of Sirtuins. Like NDR2, LATS1 can be acetylated by p300, but deacetylation takes place by distinct Sirtuins, namely SIRT3 and SIRT4 (Yang S. et al., 2022). Further, SIRT7 deacetylates the DNA damage-binding protein 1 (DDB1), which under acetylated conditions is involved in ubiquitination and degradation of LATS1 (Mo et al., 2017). While the evidence linking NDR kinases to Sirtuins is still limited, it underscores an entirely novel regulatory mechanism of NDR kinases that positions them within the central regulatory network of lifespan and longevity.

Lastly, in human tumor cells with high glucose uptake, the O-GlcNAc transferase (OGT) has been shown to O-GlcNacylate YAP and disrupt its interaction with LATS, decreasing YAP-phosphorylation and degradation, therefore increasing YAP-dependent transcription in an AMPK-independent manner. Interestingly OGT is under YAP-dependent transcription, forming another feedback loop with the involvement of NDR-kinases in nutrient signaling pathways (Peng et al., 2017).

One consequence of the increased metabolic activity of senescent cells is elevated glycogenesis through a mechanism that involves the activation of glycogen synthase, downregulation of Glycogen synthase kinase 3 beta (GSK-3β) and an increase in reactive oxygen species (ROS) (Seo et al., 2008). It has been shown that GSK-3 β signaling is altered also in the murine brain during aging, particularly in the hippocampus (Drulis-Fajdasz et al., 2018). Interestingly, in vitro it has been shown that GSK-3β also inhibits NDR1 activation, emphasizing the protective role of NDR1 in preventing cell death under increased oxidative stress (Enomoto et al., 2012). Similarly, in human melanoma cells, LATS1 has also been implicated in the regulation of ROS, and LATS1 knockdown results in increased oxidative stress (Kazimierczak et al., 2021).

NDR kinases seem to play a major role in the regulation of (macro) autophagy. Some early evidence pointed out that a loss-of-function mutation of Wts impaired autophagy and contributed to tissue degeneration. Interestingly, overexpression of the downstream effectors of Wts, Yki and scalloped (Sd), failed to rescue the effect, and that the activation of PI3K-Akt-Tor-pathway was essential for the phenotype in mutant flies (Dutta and Baehrecke, 2008), which indicated that Wts participated in autophagy regulation independently of the core Hippo signaling, through Tor signaling. Wts also plays a crucial role in autophagy-mediated cholesterol trafficking and subsequent steroid production in Drosophila. Yki-dependent transcription of the miRNA bantam enhances Tor activation and inhibits ecdysone receptor signaling, a potent inducer of autophagy in Drosophila. This process results in diminished mobilization and trafficking of cholesterol (Texada et al., 2019). In mammals, NDR1/2 enhances the inhibitory impact of the guanine nucleotide-exchange factor Rabin8 on autophagy. This effect was shown to be independent of Rabin8′s guanine-exchange-factor activity toward its downstream target RAB8 or others like SEC15 and Mammalian TRAPPII-specific subunit 130 (mTRS130) (Amagai et al., 2015). In the same study, it was observed that the silencing of NDR1/2 leads to a reduction in mTORC1 activity, but silencing Rabin8 did not have an impact on it. These observations suggest that besides Rabin8, additional mechanisms through which NDR1/2 inhibits autophagy exist, for instance by activating mTORC1 (Coyle et al., 2004; Joffre et al., 2015).

More direct recent evidence supporting the pleiotropic role of NDR kinases in the regulation of autophagy is that NDR1 interacts with BECLIN1 and other proteins that are part of the same complex required for early autophagosome formation, and silencing NDR1 in human cell lines or its homolog Trc in Drosophila impaired autophagy (Joffre et al., 2015). Interestingly, NDR1 has been demonstrated to phosphorylate the nuclear exit protein exportin1 (XPO1) at S1055, thereby influencing the nuclear exit of BECLIN1, YAP and itself (Martin et al., 2019). This discovery showcases the idea that the NDR family of kinases have multiple and sometimes opposing roles, in this case, in the regulation of autophagy. A different mechanism has been proposed for the role of NDR2 in autophagy. Under conditions of nutrient starvation, the E3-ubiquitin ligase Tripartite Motif Containing 27 (TRIM27) ubiquitinates NDR2 at L6 and L11 which enhances NDR2 activity, resulting in the downstream phosphorylation of Unc-51-like kinase 1 (ULK1) at S495. Notably, this ULK1 phosphorylation leads to increased binding of ULK1 to TRIM27 and ULK1 polyubiquitination. Consequently, this polyubiquitination promotes the enhanced proteasomal turnover of ULK1, suggesting an inhibitory role for NDR2 in autophagy initiation. Intriguingly, the frequent presence of TRIM27 overexpression in breast cancer (BC) patients is associated with tumorigenesis, potentially through the inhibition of ULK1-mediated autophagy (Yang Y. et al., 2022).

Due to its high level of similitude, it is usually assumed that NDR1 and NDR2 have complementary and sometimes overlapping functions. A notable exception is in the CNS, as it has been suggested that NDR1 expression decreases postnatally and that NDR2 is the main functional kinase of them in the adult rodent brain (Zallen et al., 2000). Recently, a double KO of Stk38 and Stk38l in mice has been linked to neurodegeneration in both adult and prenatal mice. This neurodegeneration is associated with impaired autophagy and the mechanism appears to involve the absence of NDR1/2-dependent phosphorylation of the endocytosis protein RAPH1. This deficiency leads to impaired endocytosis of the autophagy protein Autophagy-related protein (ATG) 9A at the presynapse, subsequently resulting in reduced axonal trafficking (Roşianu et al., 2023). Lastly, one notable interaction of NDR kinases and autophagy is that of the Drosophila Trc with Atg8 (Tsapras et al., 2022), which is the homolog of the Microtubule-associated proteins 1A/1B light chain 3 (LC3A/B). Although this interaction has not been described in mammals so far, it indicates that the NDR kinases are tightly associated with the regulation of autophagy and have to be considered one of the main regulators of macroautophagy. With respect to other types of autophagy, it is known that NDR1 is needed for mitochondrial clearance through mitophagy upon Extracellular matrix (ECM) detachment of Ras-transformed cells. NDR1 also participates in chaperone-assisted selective autophagy (CASA), a type of chaperone-mediated autophagy (Carra et al., 2008; Gamerdinger et al., 2009; Arndt et al., 2010; Klimek et al., 2017), by binding and inhibiting the function of the cochaperone BCL-2-associated athanogene 3 (BAG3) (Klimek et al., 2019) in tension-dependent degradation of filamins (Arndt et al., 2005, 2010). It is important to note that BAG3-mediated protein clearance is critical for the disposal of proteins associated with neurodegeneration like the AD-related protein TAU (Ji et al., 2019), Huntingtin (Klimek et al., 2017) and ALS-related Superoxide dismutase 1 (SOD1) (Crippa et al., 2010; Gamerdinger et al., 2011). This function of NDR kinases is already being explored for the treatment of age-related diseases. For instance, activating LATS1 in mice, either through the use of the traditional Chinese medicine compound Paris saponin VII or Long non-coding RNAs (lncRNAs) like RP1-59D14.5, can effectively reduce the growth of BC or prostate cancer cells in mice. This reduction is attributed to the induction of autophagy in breast and prostate cancer cells, respectively (Xiang et al., 2022; Zhong et al., 2022). Finally, the extract of the plant Radix scrophulariae has recently been shown to inhibit thyroid growth in a rat-hyperthyroidism model, through MST-LATS1-dependent autophagy activation (Zhang et al., 2023).

NDR kinases also seem to participate in protein folding by interacting with the chaperone HSP (Heat shock protein) 90, which is one of the two main chaperones involved in maintaining proteostasis through the regulation of protein folding and stabilization, particularly in the CNS. In vitro work shows, that when HSP90 is inhibited, there is a notable decrease in the levels and activity of LATS1/2 (Huntoon et al., 2010) and NDR1 (Enomoto et al., 2013). From a mechanistic standpoint, it has been proposed that the HSP90 isoform HSP90β plays a role in inhibiting the proteasomal degradation of LATS mediated by SMURF1 (Qu et al., 2023). Furthermore, the build-up of methylglyoxal (MG) in tumor cells results in increased glycation of HSP90. This glycation, in turn, disrupts the interaction between HSP90 and LATS1, causing impairments in Hippo signaling. Consequently, this disruption is associated with heightened cellular growth and an increased potential for metastasis (Nokin et al., 2016). HSP90 also plays a crucial role in the disassociation and reactivation of LATS2 protein aggregates that form during heat shock. These aggregates, in turn, induce protein-phosphatase-1-dependent dephosphorylation of LATS2, highlighting the indispensable function of HSP90 in the dynamic regulation of LATS2 in response to protein stress (Jiang et al., 2021). It is also worth noting that in yeast, the LATS1/2 homolog CBK1 has been demonstrated to modulate HSP70 and inhibit the nuclear toxicity associated with huntingtin protein aggregates (Wolfe et al., 2014). Mirroring this pro-survival mechanism, HSP70 in mammals can form a complex with BAG3 that regulates the early aggresome formation in response to the accumulation of abnormal polypeptides in a LATS1-dependent manner (Meriin et al., 2018).

In summary, it is evident that NDR kinases play a crucial role in regulating proteostasis, specifically in governing protein stability through interaction with HSP90 or HSP70, and also the eventual protein degradation through autophagy. These kinases contribute to various points in the autophagic pathway, and it is crucial to underscore that their global impact on autophagy is highly complex; depending on the specific level of the pathway at which they participate, the distinct stress conditions inducing autophagy, and the particular cell type involved. Remarkably, their importance is especially evident in neuronal proteostasis, and the disturbance of NDR kinases during aging could potentially signify a previously unrecognized aspect of age-related neurodegenerative diseases. Finally, considering the crucial role of autophagy in memory maintenance during aging (Glatigny et al., 2019), this dysregulation may contribute to cognitive decline and needs to be further explored.

Several studies have reported that the stiffness of the brain tissue changes throughout aging (Gefen et al., 2003; Sack et al., 2009; Elkin et al., 2010). This altered mechanical signaling from the extracellular space can be sensed via integrin receptors on the membrane and Hippo pathway and consequently triggers cytoskeleton remodeling as a response (Cai et al., 2021). Increased stiffness of brain tissue may cause age-related alterations such as loss of function of progenitor cells over time (Segel et al., 2019) or modulation of neuronal morphology (Si et al., 2023). The relationship between NDR kinases and ECM is tightly conserved across species. The LATS kinase homolog DBF2 in S. cerevisiae phosphorylates and activates both chitin synthase CHS2 and CYK3 during cell division. The localization of CYK3 is dependent on DBF2, setting up a mechanism for the direct control of the primary septum remodeling during the cell cycle, which is equivalent to the metazoan ECM (Oh et al., 2012). NDR kinases contribute to these processes both through the Hippo pathway and as critical regulators of integrin-mediated intracellular signaling. In this line, we have previously shown that NDR2 can modulate integrin receptor trafficking and activity in T cells (Waldt et al., 2018) and murine neurons (Rehberg et al., 2014). Furthermore, NDR kinases regulate the arborization of dendrites and axons as well as spine development and synaptic function in mammalian neurons (Ultanir et al., 2012; Rehberg et al., 2014). We showed that NDR2 controls substrate selectivity by regulating integrin subunit availability in growth cones during neurite growth (Demiray et al., 2018). Notably, integrin receptor regulation is also addressed in aging-associated pathologies, as enhancing integrin signaling holds promise in alleviating impairments in blood-brain barrier integrity in rats (Halder et al., 2023) and promoting cellular regeneration (Rozo et al., 2016; Ojha et al., 2022).

Moreover, functioning as a scaffold, the ECM supports cells, allowing them to perceive external forces and maintain their shape (Hynes, 2009) by transducing mechanical cues from the environment to the cells (Humphrey et al., 2014). Along this line, previous studies revealed a functional link between the ECM mechanotransducer glycoprotein AGRIN and Hippo pathway mediator YAP. AGRIN, as a sensor of ECM stiffness, increases the stability of YAP by the focal adhesion and LRP4/MUSK receptor pathways. AGRIN inhibits the focal adhesion assembly of Hippo pathway proteins by promoting ILK-PAK1 signaling and decreasing MERLIN and LATS1/2 interaction (Chakraborty et al., 2017). AGRIN also plays a role in supporting murine adult hippocampal neurogenesis (Zhang et al., 2019), facilitating synaptogenesis in developmental stages in a rat model of post-exercise stroke (Zhang et al., 2020), maintaining murine adult NMJs (Samuel et al., 2012), and is implicated in the pathogenesis of AD (Donahue et al., 1999; Verbeek et al., 1999). Altogether, NDR kinases could potentially be involved in AGRIN function of the developing and aging brain.

Beyond its mechanical function and role in transduction, ECM serves as a cohesive substrate for cell movement. This adhesive property is critical during cell migration and in processes such as development, wound healing, and regeneration (Rolfe and Grobbelaar, 2012; Kular et al., 2014). Fibronectin (FN), an important member of brain ECM, has a neuroprotective function in axonal regeneration and neurite outgrowth of cortical and hippocampal adult mice neurons (Tonge et al., 2012) and it diminishes during aging in the brain (Wang et al., 2011). Interestingly, it has been shown that FN adhesion increases the accumulation of YAP in the nucleus. Mechanistically, FN activates the focal adhesion kinase (FAK), which negatively regulates LATS1/2 via PI3K signaling. Reduction of LATS1/2 activity leads to YAP nuclear accumulation and transcriptional response in response to FN adhesion (Kim and Gumbiner, 2015). Notably, integrin receptors on the membrane recognize the FN in ECM and their activity can be modulated by NDR2 kinase activity (Rehberg et al., 2014), indicating the potential role of NDR Kinases in the neuroprotective functions of FN. Furthermore, cytoskeleton remodeling is a key downstream target of ECM signals and alterations in cytoskeletal dynamics have been closely associated with aging (Starodubtseva, 2011; Zahn et al., 2011; Lai and Wong, 2020). Along this line, in vitro experiments using human cell lines have shown that disturbance of microtubules with nocodazole reduces the activity of LATS1/2, while the disruption of the actin cytoskeleton with latrunculin B activates LATS1/2 (Zhao et al., 2012), implying the involvement of the cytoskeleton in the LATS-dependent regulation of YAP activity.

Taken together, the modulation of immune signaling between cells, regulation of neuronal shape and synaptic signaling, and the transduction of mechanosignalling driven by the extracellular matrix underscore the crucial role of NDR kinases in governing intercellular communication within the brain. However, further studies are necessary to specifically address the role of NDR kinases in the context of age-associated alterations in cellular communication in the nervous system.