Microtubule Affinity-regulating Kinase Research Articles

SARS-CoV-2-derived protein Orf9b enhances MARK2 activity via interaction with the autoinhibitory KA1 domain.

Microtubule affinity-regulating kinase 2 (MARK2) is a Ser/Thr protein kinase that regulates cell polarity and immune responses. Here, we report that Orf9b, one of the accessory proteins encoded in the SARS-CoV-2 genome, increases MARK2 activity via interaction with the autoinhibitory KAI domain. We found that co-expression of Orf9b enhances the kinase activity of MARK2 in HEK293 cells. Orf9b does not bind to or enhance the activity of the mutant form of MARK2 lacking the KA1 domain. Orf9b lowers inhibitory phosphorylation of MARK2 at T595 while mutation experiments indicate that this site is dispensable for Orf9b-mediated enhancement of MARK2 activity. Our results suggest that Orf9b enhances MARK2 activity by binding the autoinhibitory KA1 domain, which closely interacts with the kinase domain.

MARK2 variants cause autism spectrum disorder via the downregulation of WNT/β-catenin signaling pathway.

Microtubule affinity-regulating kinase 2 (MARK2) contributes to establishing neuronal polarity and developing dendritic spines. Although large-scale sequencing studies have associated MARK2 variants with autism spectrum disorder (ASD), the clinical features and variant spectrum in affected individuals with MARK2 variants, early developmental phenotypes in mutant human neurons, and the pathogenic mechanism underlying effects on neuronal development have remained unclear. Here, we report 31 individuals with MARK2 variants and presenting with ASD, other neurodevelopmental disorders, and distinctive facial features. Loss-of-function (LoF) variants predominate (81%) in affected individuals, while computational analysis and invitro expression assay of missense variants supported the effect of MARK2 loss. Using proband-derived and CRISPR-engineered isogenic induced pluripotent stem cells (iPSCs), we show that MARK2 loss leads to early neuronal developmental and functional deficits, including anomalous polarity and dis-organization in neural rosettes, as well as imbalanced proliferation and differentiation in neural progenitor cells (NPCs). Mark2+/- mice showed abnormal cortical formation and partition and ASD-like behavior. Through the use of RNA sequencing (RNA-seq) and lithium treatment, we link MARK2 loss to downregulation of the WNT/β-catenin signaling pathway and identify lithium as a potential drug for treating MARK2-associated ASD.

ER-phagy restrains inflammatory responses through its receptor UBAC2

ER-phagy, a selective form of autophagic degradation of endoplasmic reticulum (ER) fragments, plays an essential role in governing ER homeostasis. Dysregulation of ER-phagy is associated with the unfolded protein response (UPR), which is a major clue for evoking inflammatory diseases. However, the molecular mechanism underpinning the connection between ER-phagy and disease remains poorly defined. Here, we identified ubiquitin-associated domain-containing protein 2 (UBAC2) as a receptor for ER-phagy, while at the same time being a negative regulator of inflammatory responses. UBAC2 harbors a canonical LC3-interacting region (LIR) in its cytoplasmic domain, which binds to autophagosomal GABARAP. Upon ER-stress or autophagy activation, microtubule affinity-regulating kinase 2 (MARK2) phosphorylates UBAC2 at serine (S) 223, promoting its dimerization. Dimerized UBAC2 interacts more strongly with GABARAP, thus facilitating selective degradation of the ER. Moreover, by affecting ER-phagy, UBAC2 restrains inflammatory responses and acute ulcerative colitis (UC) in mice. Our findings indicate that ER-phagy directed by a MARK2-UBAC2 axis may provide targets for the treatment of inflammatory disease.

Function of a complex of p-Y42 RhoA GTPase and pyruvate kinase M2 in EGF signaling pathway in glioma cells.

Epidermal growth factor (EGF) is known to be a critical stimulant for inducing the proliferation of glioma cancer cells. In our study, we observed that GST-RhoA binds to pyruvate kinase M2 (PKM2) invitro. While EGF reduced the levels of RhoA protein, it significantly increased p-Y42 RhoA, as well as PKM1 and PKM2 in LN18 glioma cell line. We determined that RhoA undergoes degradation through ubiquitination involving SCF1 and Smurf1. Interestingly, we observed that p-Y42 RhoA binds to PKM2, while the dephosphomimetic form, RhoA Y42F, did not. Additionally, our observation revealed that PKM2 stabilized both RhoA and p-Y42 RhoA. Importantly, RhoA, p-Y42 RhoA, and PKM2, but not RhoA-GTP, were localized in the nucleus upon EGF stimulation. Knockdown of RhoA with siRNA resulted in the reduced levels of phosphoglycerate kinase1 (PGK1) and microtubule affinity-regulating kinase 4 (MARK). Furthermore, we found that the promoter of PGK1 was associated with β-catenin and YAP. Notably, p-Y42 RhoA and PKM2 co-immunoprecipitated with β-catenin and YAP. Based on these findings, we proposed a novel mechanism by which p-Y42 RhoA and PKM2, in conjunction with β-catenin and YAP, regulate PGK1 expression, contributing to the progression of glioma upon EGF.

α-Tocotrienol Protects Neurons by Preventing Tau Hyperphosphorylation via Inhibiting Microtubule Affinity-Regulating Kinase Activation.

In the pathological process of Alzheimer's disease, neuronal cell death is closely related to the accumulation of reactive oxygen species. Our previous studies have found that oxidative stress can activate microtubule affinity-regulating kinases, resulting in elevated phosphorylation levels of tau protein specifically at the Ser262 residue in N1E-115 cells that have been subjected to exposure to hydrogen peroxide. This process may be one of the pathogenic mechanisms of Alzheimer's disease. Vitamin E is a fat-soluble, naturally occurring antioxidant that plays a crucial role in biological systems. This study aimed to examine the probable processes that contribute to the inhibiting effect on the abnormal phosphorylation of tau protein and the neuroprotective activity of a particular type of vitamin E, α-tocotrienol. The experimental analysis revealed that α-tocotrienol showed significant neuroprotective effects in the N1E-115 cell line. Our data further suggest that one of the mechanisms underlying the neuroprotective effects of α-tocotrienol may be through the inhibition of microtubule affinity-regulated kinase activation, which significantly reduces the oxidative stress-induced aberrant elevation of p-Tau (Ser262) levels. These results indicate that α-tocotrienol may represent an intriguing strategy for treating or preventing Alzheimer's disease.

Role of MARK4 in methamphetamine-induced acute kidney injury.

Methamphetamine (METH) is an illicit psychoactive substance that can damage various organs, with the urinary system being one of its significant targets. This study aims to explore the role of microtubule affinity-regulating kinase 4 (MARK4) in METH-induced acute kidney injury (AKI). A total of 10 healthy adult male C57BL/6 mice were randomly divided into a control group and a METH group, 5 mice in each group. The METH group was administered METH (20 mg/kg, intraperitoneally, once daily for 3 consecutive days), while the control group received an equal volume of physiological saline. The mice were executed 24 hours after the final injection, and the success of the AKI model was detected by blood serum creatinine, blood urea nitrogen, and renal HE staining. Proteins differentially expressed between kidney tissues with METH-induced AKI and normal kidney tissues were screened by proteomics techniques and subjected to gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and bioinformatics analysis. The accuracy of proteomic data was validated using Western blotting, and the expression levels of MARK4 and cleaved caspase-3 in mouse kidneys were measured. We further explored the role of MARK4 in METH-induced AKI. Firstly, a METH toxicity model was established in BUMPT cells to screen the appropriate concentration and time of METH treatment; the viability of BUMPT cells after METH treatment and the expression of cleaved caspase-3 were detected by interfering with MARK4 expression through inhibitors. The proteomic analysis of kidney tissues from METH and control groups screened for a total of 17 differentially expressed proteins, of which 11 were up-regulated and 6 were down-regulated (all P<0.05). The expression levels of MARK4 and cleaved caspase-3 were elevated in the kidneys of METH-treated mice (both P<0.05). The activity of BUMPT cells gradually decreased with increasing METH treatment concentration (all P<0.05), where the viability of BUMPT cells decreased to about 60% after METH treatment at 4 mmol/L. Compared with the control group, expression levels of MARK4 and cleaved caspase-3 were increased with higher METH concentrations and longer exposure times in a concentration- and time-dependent manner (all P<0.05). Inhibition of MARK4 expression improved METH-induced decrease in BUMPT cell activity, down-regulated the expression of cleaved caspase-3, and decreased the apoptosis of BUMPT cells induced by METH. MARK4 is highly expressed in a mouse model of METH-induced AKI, and MARK4 mediates METH-induced AKI by regulating cell apoptosis.

Vanillin-Isatin Hybrid-Induced MARK4 Inhibition As a Promising Therapeutic Strategy against Hepatocellular Carcinoma.

Microtubule affinity-regulating kinase 4 (MARK4) is a serine-threonine kinase that phosphorylates microtubule-associated proteins (MAPs) and increases the microtubule dynamics. Due to its direct involvement in initiation, cell division, progression, and cancer metastasis, MARK4 is considered a potential therapeutic target. Here, we designed, synthesized, and characterized vanillin-isatin hybrids and evaluated their MARK4 inhibitory potential. All of the compounds strongly bind to MARK4 and interact closely with the active site residues. Finally, the compound VI-9 was selected for further investigation due to its high binding affinity and strong MARK4 inhibitory potential. Tau-phosphorylation assay has further confirmed that VI-9 significantly reduced the activity of MARK4. Compared with vanillin, VI-9 showed a better binding affinity and MARK4 inhibitory potential. Cell viability assays on human hepatocellular carcinoma (HCC) cell lines C3A and SNU-475 revealed that VI-9 inhibited their growth and proliferation. In addition, these compounds were nontoxic (up to 200 μM) for noncancerous (HEK-293) cells. Interestingly, VI-9 induces apoptosis and decreases the metastatic potential of the C3A and SNU-475 cell lines. The present work opens a newer avenue for vanillin-isatin hybrids and their derivatives in developing MARK4-targeted anticancer therapies.

Navigating the Maze of Kinases: CaMK-like Family Protein Kinases and Their Role in Atherosclerosis.

Circulating low-density lipoprotein (LDL) levels are a major risk factor for cardiovascular diseases (CVD), and even though current treatment strategies focusing on lowering lipid levels are effective, CVD remains the primary cause of death worldwide. Atherosclerosis is the major cause of CVD and is a chronic inflammatory condition in which various cell types and protein kinases play a crucial role. However, the underlying mechanisms of atherosclerosis are not entirely understood yet. Notably, protein kinases are highly druggable targets and represent, therefore, a novel way to target atherosclerosis. In this review, the potential role of the calcium/calmodulin-dependent protein kinase-like (CaMKL) family and its role in atherosclerosis will be discussed. This family consists of 12 subfamilies, among which are the well-described and conserved liver kinase B1 (LKB1) and 5' adenosine monophosphate-activated protein kinase (AMPK) subfamilies. Interestingly, LKB1 plays a key role and is considered a master kinase within the CaMKL family. It has been shown that LKB1 signaling leads to atheroprotective effects, while, for example, members of the microtubule affinity-regulating kinase (MARK) subfamily have been described to aggravate atherosclerosis development. These observations highlight the importance of studying kinases and their signaling pathways in atherosclerosis, bringing us a step closer to unraveling the underlying mechanisms of atherosclerosis.

Ropinirole reverses the effects of neuroinflammation, and cellular demise by downregulating the MARK4-NFκβ signaling system in Alzheimer's disease

Ropinirole (ROP) is a dopamine agonist that can cross the blood-brain barrier (BBB), which is crucial for drugs targeting neurological conditions like Alzheimer's disease (AD). The rationale for the current research is to investigate the potential of ROP as an inhibitor of Microtubule affinity regulating kinase 4 (MARK4)-NFκβ in neurodegenerative diseases, specifically AD. The interaction between ROP and MARK4-NFκβ holds significant promise in the realm of drug discovery and therapeutic interventions for diseases like AD. Molecular docking and biophysical characterization demonstrate how ROP effectively hinders MARK4 activity, offering detailed insights into their molecular interactions. The present research also investigates the biological aspect of MARK4 shows promise in treating AD, with neuroinflammation playing a crucial role in the disease's progression. Aβ42 and ROP were co-administered directly into the cells for the establishment of the AD model. We confirmed that ROP can inhibit the path of MARK4 activity, as evidenced by biophysical characterization, and can enhance the cell viability, lowers the expression of MARK4, decrease the rate of oxidative stress, and attenuate the expression of NFκβ, leading to reduced neuronal apoptosis in an in vitro-induced Aβ model. Overall, this research provides valuable mechanistic insights into the neuroprotective potential of ROP and its ability to target the MARK4-NFκβ pathway.

MicroRNA miR-7-5p targets MARK2 to control metamorphosis in Galeruca daurica

The MARK2 gene, coding microtubule affinity-regulating kinase or serine/threonine protein kinase, is an important modulator in organism microtubule generation and cell polarity. However, its role in the metamorphosis of insects remains unknown. In this study, we found a conserved miRNA, miR-7-5p, which targets MARK2 to participate in the regulation of the larval-pupal metamorphosis in Galeruca daurica. The dual luciferase reporter assay showed that miR-7-5p interacted with the 3’ UTR of MARK2 and repressed its expression. The expression profiling of miR-7-5p and MARK2 displayed an opposite trend during the larval-adult development process. In in-vivo experiments, overexpression of miR-7-5p by injecting miR-7-5p agomir in the final instar larvae down-regulated MARK2 and up-regulated main ecdysone signaling pathway genes including E74, E75, ECR, FTZ-F1 and HR3, which was similar to the results from knockdown of MARK2 by RNAi. In contrast, repression of miR-7-5p by injecting miR-7-5p antagomir obtained opposite effects. Notably, both overexpression and repression of miR-7-5p in the final instar larvae caused abnormal molting and high mortality during the larval-pupal transition, and high mortality during the pupal-adult transition. The 20-hydroxyecdysone (20E) injection experiment showed that 20E up-regulated miR-7-5p whereas down-regulated MARK2. This study reveals that the accurate regulation of miRNAs and their target genes is indispensable for insect metamorphosis.

MARK1 regulates dendritic spine morphogenesis and cognitive functions in vivo

Dendritic spines play a pivotal role in synaptic communication and are crucial for learning and memory processes. Abnormalities in spine morphology and plasticity are observed in neurodevelopmental and neuropsychiatric disorders, yet the underlying signaling mechanisms remain poorly understood. The microtubule affinity regulating kinase 1 (MARK1) has been implicated in neurodevelopmental disorders, and the MARK1 gene shows accelerated evolution in the human lineage suggesting a role in cognition. However, the in vivo role of MARK1 in synaptogenesis and cognitive functions remains unknown. Here we show that forebrain-specific conditional knockout (cKO) of Mark1 in mice causes defects in dendritic spine morphogenesis in hippocampal CA1 pyramidal neurons with a significant reduction in spine density. In addition, we found loss of MARK1 causes synaptic accumulation of GKAP and GluA2. Furthermore, we found that MARK1 cKO mice show defects in spatial learning in the Morris water maze and reduced anxiety-like behaviors in the elevated plus maze. Taken together, our data show a novel role for MARK1 in regulating dendritic spine morphogenesis and cognitive functions in vivo.

A novel role for microtubule affinity-regulating kinases in neuropathic pain.

Neuropathic pain affects millions of patients, but there are currently few viable therapeutic options available. Microtubule affinity-regulating kinases (MARKs) regulate the dynamics of microtubules and participate in synaptic remodelling. It is unclear whether these changes are involved in the central sensitization of neuropathic pain. This study examined the role of MARK1 or MARK2 in regulating neurosynaptic plasticity induced by neuropathic pain. A rat spinal nerve ligation (SNL) model was established to induce neuropathic pain. The role of MARKs in nociceptive regulation was assessed by genetically knocking down MARK1 or MARK2 in amygdala and systemic administration of PCC0105003, a novel small molecule MARK inhibitor. Cognitive function, anxiety-like behaviours and motor coordination capability were also examined in SNL rats. Synaptic remodelling-associated signalling changes were detected with electrophysiological recording, Golgi-Cox staining, western blotting and qRT-PCR. MARK1 and MARK2 expression levels in amygdala and spinal dorsal horn were elevated in SNL rats. MARK1 or MARK2 knockdown in amygdala and PCC0105003 treatment partially attenuated pain-like behaviours along with improving cognitive deficit, anxiogenic-like behaviours and motor coordination in SNL rats. Inhibition of MARKs signalling reversed synaptic plasticity at the functional and structural levels by suppressing NR2B/GluR1 and EB3/Drebrin signalling pathways both in amygdala and spinal dorsal horn. These results suggest that MARKs-mediated synaptic remodelling plays a key role in the pathogenesis of neuropathic pain and that pharmacological inhibitors of MARKs such as PCC0105003 could represent a novel therapeutic strategy for the management of neuropathic pain.

Harnessing memantine in Alzheimer's disease therapy through inhibition of microtubule affinity-regulating kinase: Mechanistic insights

Alzheimer's disease (AD) is one of the neurodegenerative disorder that primarily affects memory, thinking, and behavior, eventually leading to severe cognitive impairment. Therapeutic management of AD is urgently needed to improve the quality and lifestyle of patients. Tau phosphorylating kinases are considered attractive therapeutic targets. Microtubule affinity-regulating kinase 4 (MARK4) is directly linked with pathological phosphorylations of tau, highlighting its role in the therapeutic targeting of AD. The current manuscript shows the MARK4 inhibitory effect of Memantine (MEM), a drug used in treating AD. We have performed fluorescence based binding measurements, enzyme inhibition assay, docking and molecular dynamics (MD) simulations to understand the binding of of MARK4 and MEM and subsequent inhibition in the kinase activity. A 100 ns MD simulations provided a detailed analysis of MARK4-MEM complex and the role of potential critical residues in the binding. Finally, this study provides molecular insights into the therapeutic implication of MEM in AD therapeutics. We propose MEM effectively inhibits MARK4, it may be implicated in the development of targeted and efficient treatments for AD.

Microtubule-affinity regulating kinase family members distinctively affect tau phosphorylation and promote its toxicity in a Drosophila model.

Accumulation of abnormally phosphorylated tau and its aggregation constitute a significant hallmark of Alzheimer's disease (AD). Tau phosphorylation at Ser262 and Ser356 in the KXGS motifs of microtubule-binding repeats plays a critical role in its physiological function and AD disease progression. Major tau kinases to phosphorylate tau at Ser262 and Ser356 belong to the Microtubule Affinity Regulating Kinase family (MARK1-4), which are considered one of the major contributors to tau abnormalities in AD. However, whether and how each member affects tau toxicity in vivo is unclear. We used transgenic Drosophila as a model to compare the effect on tau-induced neurodegeneration among MARKs in vivo. MARK4 specifically promotes tau accumulation and Ser396 phosphorylation, which yields more tau toxicity than was caused by other MARKs. Interestingly, MARK1, 2, and 4 increased tau phosphorylation at Ser262 and Ser356, but only MARK4 caused tau accumulation, indicating that these sites alone did not cause pathological tau accumulation. Our results revealed MARKs are different in their effect on tau toxicity, and also in tau phosphorylation at pathological sites other than Ser262 and Ser356. Understanding the implementation of each MARK into neurodegenerative disease helps to develop more target and safety therapies to overcome AD and related tauopathies.

Interaction of Sp1 and Setd8 promotes vascular smooth muscle cells apoptosis by activating Mark4 in vascular calcification.

Vascular calcification (VC) is directly related to high mortality in chronic kidney disease (CKD), and cellular apoptosis of vascular smooth muscle cells (VSMCs) is a crucial process in the initiation of VC. Microtubule affinity-regulating kinase 4 (Mark4), known as a serine/threonine protein kinase, can induce cell apoptosis and autophagy by modulating Akt phosphorylation. However, the potential functions and molecular mechanisms of Mark4 in VSMCs apoptosis and calcification need to be further explored. Initially, our data indicated that the mRNA expression of Mark4 was prominently elevated in high phosphorus-stimulated human VSMCs compared with the other members in Marks. Consistently, Mark4 expression was found to be significantly increased in the calcified arteries of both CKD patients and rats. In vitro, silencing Mark4 suppressed apoptosis-specific marker expression by promoting Akt phosphorylation, finally attenuating VSMCs calcification induced by high phosphate. Mechanically, the transcription factor Sp1 was enriched in the Mark4 promoter region and modulated Mark4 transcription. Moreover, SET domain-containing protein 8 (Setd8) was proved to interact with Sp1 and jointly participated in the transcriptional regulation of Mark4. Finally, rescue experiments revealed that Setd8 contributed to VSMCs apoptosis and calcification by modulating Mark4 expression. In conclusion, these findings reveal that Mark4 is transcriptionally activated by Sp1, which is interacted with Setd8, to promote VSMCs calcification through Akt-mediated antiapoptotic effects, suggesting that Mark4 represents a potent and promising therapeutic target for VC in CKD.

Mark4 ablation attenuates pathological phenotypes in a mouse model of tauopathy.

Accumulation of abnormally phosphorylated tau proteins is linked to various neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia. Microtubule affinity-regulating kinase 4 (MARK4) has been genetically and pathologically associated with Alzheimer's disease and reported to enhance tau phosphorylation and toxicity in Drosophila and mouse traumatic brain-injury models but not in mammalian tauopathy models. To investigate the role of MARK4 in tau-mediated neuropathology, we crossed P301S tauopathy model (PS19) and Mark4 knockout mice. We performed behaviour, biochemical and histology analyses to evaluate changes in PS19 pathological phenotype with and without Mark4. Here, we demonstrated that Mark4 deletion ameliorated the tau pathology in a mouse model of tauopathy. In particular, we found that PS19 with Mark4 knockout showed improved mortality and memory compared with those bearing an intact Mark4 gene. These phenotypes were accompanied by reduced neurodegeneration and astrogliosis in response to the reduction of pathological forms of tau, such as those phosphorylated at Ser356, AT8-positive tau and thioflavin S-positive tau. Our data indicate that MARK4 critically contributes to tau-mediated neuropathology, suggesting that MARK4 inhibition may serve as a therapeutic avenue for tauopathies.

Studying Microtubule Dynamics in Human Neurons: Two-Dimensional Microtubule Tracing and Kymographs in iPSC- and SH-SY5Y-Derived Neurons for Tau Research.

The study of microtubule (MT) dynamics is essential for the understanding of cellular transport, cell polarity, axon formation, and other neurodevelopmental mechanisms. All these processes rely on the constant transition between assembly and disassembly of tubulin polymers to/from MTs, known as dynamic instability. This process is well-regulated, among others, by phosphorylation of microtubule-associated proteins (MAP), including the Tau protein. Protein kinases, in particular the microtubule affinity regulating kinase (MARK), regulate the MT-Tau interaction, inducingTau dissociation by phosphorylation. Phosphorylated Tau dissociates from microtubules forming insoluble aggregates known as neurofibrillary tangles. These accumulations of hyperphosphorylated Tau in the neurons disrupt the physiological MT-based transport machinery within the cell and can potentially lead to the development of neurodegenerative disorders, such as Alzheimer's disease (AD) and related tauopathies. Further investigations on the MT cytoskeleton dynamics are essential as they may elucidate pathomechanisms of neurodegenerative diseases - particularly tauopathies - as well as fundamental neurodevelopmental processes.The study of thedynamic assembly and disassembly of the MT network requires live-cell imaging rather than conventional immunocytochemistry based on fixed samples. To investigate MT dynamics, we perform live-cell imaging of neurons transfected with a fluorescently tagged version of the microtubule plus-end tracking protein (+TIP) EB3. This protein associates with the growing ends of MTs and thus visualizes MT growth in real time. Our imaging analysis protocol allows the determination of quantity, orientation, and velocity of MT growth in the soma and neurites of transfected neurons,using ImageJ-based tracking software and kymographs. Furthermore, functional effects of Tau and MARK kinases on the MT cytoskeleton can be assessed by overexpression or downregulation experiments of the respective protein prior to the live imaging assay. We use two different human neuronal cell models, naive and differentiated SH-SY5Y neuroblastoma cells, and neurons derived from induced pluripotent stem cells (iPSCs), both of which have shown success as models to study Tau-related pathologies.This protocol describes an optimized method for analysis of microtubule dynamics using fluorescent tagged EB3 protein as microtubule plus end marker. In this chapter, we outline the process of neuronal transfection, live-cell imaging, and necessary time-lapse image analysis based on ImageJ in two human-derived neuronal systems, which are suitable for the analysis of Tau trafficking and sorting studies.

Gain-of-function MARK4 variant associates with pediatric neurodevelopmental disorder and dysmorphism

Microtubule affinity regulating kinase 4 (MARK4) is a serine/threonine kinase that plays a key role in tau phosphorylation and regulation of the mTOR pathway. Abnormal tau phosphorylation and dysregulation of the mTOR pathway are implicated in neurodegenerative and neurodevelopmental disorders. Here, we report a gain-of-function variant in MARK4 in two siblings with childhood-onset neurodevelopmental disability and dysmorphic features. The siblings carry a germline heterozygous missense MARK4 variant c.604T>C; p.Phe202Leu, located in the catalytic domain of the kinase, which they inherited from their unaffected, somatic mosaic mother. Functional studies show that this amino acid substitution has no impact on protein expression but instead increases the ability of MARK4 to phosphorylate tau isoforms found in the fetal and adult brain. The MARK4 variant also increases phosphorylation of ribosomal protein S6, indicating upregulation of the mTORC1 pathway. In this study, we link a germline monoallelic MARK4 variant to a childhood-onset neurodevelopmental disorder characterized by global developmental delay, intellectual disability, behavioral abnormalities, and dysmorphic features.

A cell-penetrating peptide derived from SARS-CoV-2 protein Orf9b allosterically inhibits MARK4 activity and mitigates tau toxicity

Abnormal activation of microtubule affinity-regulating kinase 4 (MARK4) and its phosphorylation of the microtubule-associated protein tau are believed to play a role in the pathogenesis of Alzheimer's disease, and MARK4 inhibition can be a strategy to develop disease-modifying therapy. Here we report the development of a membrane-permeable peptide that inhibits MARK4 activity in an allosteric manner. The SARS-CoV-2-derived protein Orf9b inhibited MARK4-mediated tau phosphorylation in primary neurons and Drosophila. Orf9b inhibited MARK4 activity in an allosteric manner and did not inhibit the activity of MARK2, which is another MARK family member and is closely related to MARK4. Co-expression of Orf9b in the fly retina expressing human tau and MARK4 suppressed phosphorylation of tau at the microtubule-binding repeats and tau-induced neurodegeneration. We identified the minimal sequence of Orf9b required to suppress MARK4 activity and fused it to a cell-permeable sequence (TAT-Orf9b10-18_78–95). Extracellular supplementation of TAT-Orf9b10-18_78–95 inhibited MARK4 activity in primary neurons, and feeding TAT-Orf9b10-18_78–95 to a fly model of tauopathy lowered phospho-tau levels and suppressed neurodegeneration. These results suggest that TAT-Orf9b10-18_78–95 is a unique class of MARK4 inhibitor and can be used to modify tau toxicity.

SPTAN1/NUMB axis senses cell density to restrain cell growth and oncogenesis through Hippo signaling.

The loss of contact inhibition is a key step during carcinogenesis. The Hippo-Yes-associated protein (Hippo/YAP) pathway is an important regulator of cell growth in a cell density-dependent manner. However, how Hippo signaling senses cell density in this context remains elusive. Here, we report that high cell density induced the phosphorylation of spectrin α chain, nonerythrocytic 1 (SPTAN1), a plasma membrane-stabilizing protein, to recruit NUMB endocytic adaptor protein isoforms 1 and 2 (NUMB1/2), which further sequestered microtubule affinity-regulating kinases (MARKs) in the plasma membrane and rendered them inaccessible for phosphorylation and inhibition of the Hippo kinases sterile 20-like kinases MST1 and MST2 (MST1/2). WW45 interaction with MST1/2 was thereby enhanced, resulting in the activation of Hippo signaling to block YAP activity for cell contact inhibition. Importantly, low cell density led to SPTAN1 dephosphorylation and NUMB cytoplasmic location, along with MST1/2 inhibition and, consequently, YAP activation. Moreover, double KO of NUMB and WW45 in the liver led to appreciable organ enlargement and rapid tumorigenesis. Interestingly, NUMB isoforms 3 and 4, which have a truncated phosphotyrosine-binding (PTB) domain and are thus unable to interact with phosphorylated SPTAN1 and activate MST1/2, were selectively upregulated in liver cancer, which correlated with YAP activation. We have thus revealed a SPTAN1/NUMB1/2 axis that acts as a cell density sensor to restrain cell growth and oncogenesis by coupling external cell-cell contact signals to intracellular Hippo signaling.