Kidins220 sets the threshold for survival of neural stem cells and progenitors to sustain adult neurogenesis.

In the adult mammalian brain, neural stem cells (NSCs) located in highly restricted niches sustain the generation of new neurons that integrate into existing circuits. A reduction in adult neurogenesis is linked to ageing and neurodegeneration, whereas dysregulation of proliferation and survival of NSCs have been hypothesized to be at the origin of glioma. Thus, unravelling the molecular underpinnings of the regulated activation that NSCs must undergo to proliferate and generate new progeny is of considerable relevance. Current research has identified cues promoting or restraining NSCs activation. Yet, whether NSCs depend on external signals to survive or if intrinsic factors establish a threshold for sustaining their viability remains elusive, even if this knowledge could involve potential for devising novel therapeutic strategies. Kidins220 (Kinase D-interacting substrate of 220 kDa) is an essential effector of crucial pathways for neuronal survival and differentiation. It is dramatically altered in cancer and in neurological and neurodegenerative disorders, emerging as a regulatory molecule with important functions in human disease. Herein, we discover severe neurogenic deficits and hippocampal-based spatial memory defects accompanied by increased neuroblast death and high loss of newly formed neurons in Kidins220 deficient mice. Mechanistically, we demonstrate that Kidins220-dependent activation of AKT in response to EGF restraints GSK3 activity preventing NSCs apoptosis. We also show that NSCs with Kidins220 can survive with lower concentrations of EGF than the ones lacking this molecule. Hence, Kidins220 levels set a molecular threshold for survival in response to mitogens, allowing adult NSCs growth and expansion. Our study identifies Kidins220 as a key player for sensing the availability of growth factors to sustain adult neurogenesis, uncovering a molecular link that may help paving the way towards neurorepair.

E2A Modulates Stemness, Metastasis, and Therapeutic Resistance of Breast Cancer.

Cancer stem cells (CSC) are considered responsible for tumor initiation, therapeutic resistance, and metastasis. A comprehensive knowledge of the mechanisms governing the acquisition and maintenance of cancer stemness is crucial for the development of new therapeutic approaches in oncology. E2A basic helix-loop-helix (bHLH) transcription factors are associated with epithelial-mesenchymal transition (EMT) and tumor progression, but knowledge of their functional contributions to cancer biology is still limited. Using a combination of in vivo and in vitro analyses in a novel PyMT-E2A conditional knockout mouse model and derived primary tumor cell lines, we report here an essential role of E2A in stemness, metastasis, and therapeutic resistance in breast cancer. Targeted deletion of E2A in the mammary gland impaired tumor-initiating ability and dedifferentiation potential and severely compromised metastatic competence of PyMT-driven mammary tumors. Mechanistic studies in PyMT-derived cell lines indicated that E2A actions are mediated by the upregulation of Snai1 transcription. Importantly, high E2A and SNAIL1 expression occurred in aggressive human basal-like breast carcinomas, highlighting the relevance of the E2A-Snail1 axis in metastatic breast cancer. In addition, E2A factors contributed to the maintenance of genomic integrity and resistance to PARP inhibitors in PyMT and human triple-negative breast cancer cells. Collectively, these results support the potential for E2A transcription factors as novel targets worthy of translational consideration in breast cancer. SIGNIFICANCE: These findings identify key functions of E2A factors in breast cancer cell stemness, metastasis, and drug resistance, supporting a therapeutic vulnerability to targeting E2A proteins in breast cancer.

Kidins220 deficiency causes ventriculomegaly via SNX27-retromer-dependent AQP4 degradation.

Several psychiatric, neurologic and neurodegenerative disorders present increased brain ventricles volume, being hydrocephalus the disease with the major manifestation of ventriculomegaly caused by the accumulation of high amounts of cerebrospinal fluid (CSF). The molecules and pathomechanisms underlying cerebral ventricular enlargement are widely unknown. Kinase D interacting substrate of 220 kDa (KIDINS220) gene has been recently associated with schizophrenia and with a novel syndrome characterized by spastic paraplegia, intellectual disability, nystagmus and obesity (SINO syndrome), diseases frequently occurring with ventriculomegaly. Here we show that Kidins220, a transmembrane protein effector of various key neuronal signalling pathways, is a critical regulator of CSF homeostasis. We observe that both KIDINS220 and the water channel aquaporin-4 (AQP4) are markedly downregulated at the ventricular ependymal lining of idiopathic normal pressure hydrocephalus (iNPH) patients. We also find that Kidins220 deficient mice develop ventriculomegaly accompanied by water dyshomeostasis and loss of AQP4 in the brain ventricular ependymal layer and astrocytes. Kidins220 is a known cargo of the SNX27-retromer, a complex that redirects endocytosed plasma membrane proteins (cargos) back to the cell surface, thus avoiding their targeting to lysosomes for degradation. Mechanistically, we show that AQP4 is a novel cargo of the SNX27-retromer and that Kidins220 deficiency promotes a striking and unexpected downregulation of the SNX27-retromer that results in AQP4 lysosomal degradation. Accordingly, SNX27 silencing decreases AQP4 levels in wild-type astrocytes whereas SNX27 overexpression restores AQP4 content in Kidins220 deficient astrocytes. Together our data suggest that the KIDINS220-SNX27-retromer-AQP4 pathway is involved in human ventriculomegaly and open novel therapeutic perspectives.

Excitotoxic targeting of Kidins220 to the Golgi apparatus precedes calpain cleavage of Rap1-activation complexes.

Excitotoxic neuronal death induced by high concentrations of glutamate is a pathological event common to multiple acute or chronic neurodegenerative diseases. Excitotoxicity is mediated through overactivation of the N-Methyl-D-aspartate type of ionotropic glutamate receptors (NMDARs). Physiological stimulation of NMDARs triggers their endocytosis from the neuronal surface, inducing synaptic activity and survival. However almost nothing is known about the internalization of overactivated NMDARs and their interacting proteins, and how this endocytic process is connected with neuronal death has been poorly explored. Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is a component of NMDAR complexes essential for neuronal viability by the control of ERK activation. Here we have investigated Kidins220 endocytosis induced by NMDAR overstimulation and the participation of this internalization step in the molecular mechanisms of excitotoxicity. We show that excitotoxicity induces Kidins220 and GluN1 traffic to the Golgi apparatus (GA) before Kidins220 is degraded by the protease calpain. We also find that excitotoxicity triggers an early activation of Rap1-GTPase followed by its inactivation. Kidins220 excitotoxic endocytosis and subsequent calpain-mediated downregulation governs this late inactivation of Rap1 that is associated to decreases in ERK activity preceding neuronal death. Furthermore, we identify the molecular mechanisms involved in the excitotoxic shutoff of Kidins220/Rap1/ERK prosurvival cascade that depends on calpain processing of Rap1-activation complexes. Our data fit in a model where Kidins220 targeting to the GA during early excitotoxicity would facilitate Rap1 activation and subsequent stimulation of ERK. At later times, activation of Golgi-associated calpain, would promote the degradation of GA-targeted Kidins220 and two additional components of the specific Rap1 activation complex, PDZ-GEF1, and S-SCAM. In this way, late excitotoxicity would turn off Rap1/ERK cascade and compromise neuronal survival.

Lysyl Oxidase-like Protein LOXL2 Promotes Lung Metastasis of Breast Cancer.

The lysyl oxidase-like protein LOXL2 has been suggested to contribute to tumor progression and metastasis, but in vivo evidence has been lacking. Here we provide functional evidence that LOXL2 is a key driver of breast cancer metastasis in two conditional transgenic mouse models of PyMT-induced breast cancer. LOXL2 ablation in mammary tumor cells dramatically decreased lung metastasis, whereas LOXL2 overexpression promoted metastatic tumor growth. LOXL2 depletion or overexpression in tumor cells does not affect extracellular matrix stiffness or organization in primary and metastatic tumors, implying a function for LOXL2 independent of its conventional role in extracellular matrix remodeling. In support of this likelihood, cellular and molecular analyses revealed an association of LOXL2 action with elevated levels of the EMT regulatory transcription factor Snail1 and expression of several cytokines that promote premetastatic niche formation. Taken together, our findings established a pathophysiologic role and new function for LOXL2 in breast cancer metastasis. Cancer Res; 77(21); 5846-59. ©2017 AACR.

Kidins220 accumulates with tau in human Alzheimer's disease and related models: modulation of its calpain-processing by GSK3ß/PP1 imbalance.

Failures in neurotrophic support and signalling play key roles in Alzheimer's disease (AD) pathogenesis. We previously demonstrated that downregulation of the neurotrophin effector Kinase D interacting substrate (Kidins220) by excitotoxicity and cerebral ischaemia contributed to neuronal death. This downregulation, triggered through overactivation of N-methyl-D-aspartate receptors (NMDARs), involved proteolysis of Kidins220 by calpain and transcriptional inhibition. As excitotoxicity is at the basis of AD aetiology, we hypothesized that Kidins220 might also be downregulated in this disease. Unexpectedly, Kidins220 is augmented in necropsies from AD patients where it accumulates with hyperphosphorylated tau. This increase correlates with enhanced Kidins220 resistance to calpain processing but no higher gene transcription. Using AD brain necropsies, glycogen synthase kinase 3-ß (GSK3ß)-transgenic mice and cell models of AD-related neurodegeneration, we show that GSK3ß phosphorylation decreases Kidins220 susceptibility to calpain proteolysis, while protein phosphatase 1 (PP1) action has the opposite effect. As altered activities of GSK3ß and phosphatases are involved in tau aggregation and constitute hallmarks in AD, a GSK3ß/PP1 imbalance may also contribute to Kidins220 decreased clearance, accumulation and hampered neurotrophin signalling from early stages of the disease pathogenesis. These results encourage searches for mutations in Kidins220 gene and their possible associations to dementias. Finally, our data support a model where the effects of excitotoxicity drastically differ when occurring in cerebral ischaemia versus progressively sustained toxicity along AD progression. The striking differences in Kidins220 stability resulting from chronic versus acute brain damage may also have important implications for the therapeutic intervention of neurodegenerative disorders.

The neuronal protein Kidins220/ARMS associates with ICAM-3 and other uropod components and regulates T-cell motility.

Kinase D interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is a protein that is mainly expressed in brain and neural cells where its function is only starting to be characterized. Here, we show that Kidins220/ARMS is also expressed in T lymphocytes where it is highly concentrated at the uropod of polarized T cells. In this cellular model, Kidins220/ARMS colocalizes with typical uropod T-cell molecules and coimmunoprecipitates with ICAM-3. Furthermore, Kidins220/ARMS associates with raft domains at the uropod and coimmunoprecipitates with caveolin-1, a molecule we show here to be also expressed in T cells. Importantly, induction of morphological polarization in primary T lymphocytes and Jurkat cells enhances Kidins220/ARMS colocalization with ICAM-3. Conversely, disruption of cell polarity provokes Kidins220/ARMS redistribution from the uropod to other cellular regions and drastically impairs its association with ICAM-3 in a protein kinase C-dependent manner. Finally, Kidins220/ARMS knockdown in human polarized T-cell lines promotes both basal and stromal cell-derived factor-1α-induced directed migration, identifying a novel function for this molecule. Altogether, our findings show that Kidins220/ARMS is a novel component of the uropod involved in the regulation of T-cell motility, an essential process for the immune response.

Kidins220/ARMS downregulation by excitotoxic activation of NMDARs reveals its involvement in neuronal survival and death pathways.

Functional and protein interactions between the N-methyl-D-aspartate type of glutamate receptor (NMDAR) and neurotrophin or ephrin receptors play essential roles in neuronal survival and differentiation. A shared downstream effector for neurotrophin- and ephrin-receptor signaling is kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS). Because this molecule is obligatory for neurotrophin-induced differentiation, we investigated whether Kidins220/ARMS and NMDAR functions were related. Here, we identify an association between these proteins and discover that excitotoxicity, a specific form of neuronal death induced by NMDAR overstimulation, dramatically decreases Kidins220/ARMS levels in cortical neurons and in a model of cerebral ischemia. Kidins220/ARMS downregulation is triggered by overactivation of NMDARs containing NR2B subunits and subsequent Ca(2+) influx, and involves a dual mechanism: rapid cleavage by the Ca(2+)-dependent protease calpain and calpain-independent silencing of Kidins220/Arms gene transcription. Additionally, Kidins220/ARMS knockdown decreases ERK activation and basal neuronal viability, and enhances neuronal death under excitotoxic conditions. Our results demonstrate Kidins220/ARMS participation in neuronal life and death pathways, and constitute the first report of its regulation under pathological conditions.

Protein kinase D intracellular localization and activity control kinase D-interacting substrate of 220-kDa traffic through a postsynaptic density-95/discs large/zonula occludens-1-binding motif.

Protein kinase D (PKD) controls protein traffic from the trans-Golgi network (TGN) to the plasma membrane of epithelial cells in an isoform-specific manner. However, whether the different PKD isoforms could be selectively regulating the traffic of their specific substrates remains unexplored. We identified the C terminus of the different PKDs that constitutes a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif in PKD1 and PKD2, but not in PKD3, to be responsible for the differential control of kinase D-interacting substrate of 220-kDa (Kidins220) surface localization, a neural membrane protein identified as the first substrate of PKD1. A kinase-inactive mutant of PKD3 is only able to alter the localization of Kidins220 at the plasma membrane when its C terminus has been substituted by the PDZ-binding motif of PKD1 or PKD2. This isoform-specific regulation of Kidins220 transport might not be due to differences among kinase activity or substrate selectivity of the PKD isoenzymes but more to the adaptors bound to their unique C terminus. Furthermore, by mutating the autophosphorylation site Ser(916), located at the critical position -2 of the PDZ-binding domain within PKD1, or by phorbol ester stimulation, we demonstrate that the phosphorylation of this residue is crucial for Kidins220-regulated transport. We also discovered that Ser(916) trans-phosphorylation takes place among PKD1 molecules. Finally, we demonstrate that PKD1 association to intracellular membranes is critical to control Kidins220 traffic. Our findings reveal the molecular mechanism by which PKD localization and activity control the traffic of Kidins220, most likely by modulating the recruitment of PDZ proteins in an isoform-specific and phosphorylation-dependent manner.