Checking NEKs: Overcoming a Bottleneck in Human Diseases

In previous years, several kinases, such as phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), and extracellular-signal-regulated kinase (ERK), have been linked to important human diseases, although some kinase families remain neglected in terms of research, hiding their relevance to therapeutic approaches. Here, a review regarding the NEK family is presented, shedding light on important information related to NEKs and human diseases. NEKs are a large group of homologous kinases with related functions and structures that participate in several cellular processes such as the cell cycle, cell division, cilia formation, and the DNA damage response. The review of the literature points to the pivotal participation of NEKs in important human diseases, like different types of cancer, diabetes, ciliopathies and central nervous system related and inflammatory-related diseases. The different known regulatory molecular mechanisms specific to each NEK are also presented, relating to their involvement in different diseases. In addition, important information about NEKs remains to be elucidated and is highlighted in this review, showing the need for other studies and research regarding this kinase family. Therefore, the NEK family represents an important group of kinases with potential applications in the therapy of human diseases

NEK6 Regulates Redox Balance and DNA Damage Response in DU-145 Prostate Cancer Cells

NEK6 is a central kinase in developing castration-resistant prostate cancer (CRPC). However, the pathways regulated by NEK6 in CRPC are still unclear. Cancer cells have high reactive oxygen species (ROS) levels and easily adapt to this circumstance and avoid cell death by increasing antioxidant defenses. We knocked out the NEK6 gene and evaluated the redox state and DNA damage response in DU-145 cells. The knockout of NEK6 decreases the clonogenic capacity, proliferation, cell viability, and mitochondrial activity. Targeting the NEK6 gene increases the level of intracellular ROS; decreases the expression of antioxidant defenses (SOD1, SOD2, and PRDX3); increases JNK phosphorylation, a stress-responsive kinase; and increases DNA damage markers (p-ATM and γH2AX). The exogenous overexpression of NEK6 also increases the expression of these same antioxidant defenses and decreases γH2AX. The depletion of NEK6 also induces cell death by apoptosis and reduces the antiapoptotic Bcl-2 protein. NEK6-lacking cells have more sensitivity to cisplatin. Additionally, NEK6 regulates the nuclear localization of NF-κB2, suggesting NEK6 may regulate NF-κB2 activity. Therefore, NEK6 alters the redox balance, regulates the expression of antioxidant proteins and DNA damage, and its absence induces the death of DU-145 cells. NEK6 inhibition may be a new strategy for CRPC therapy

On Broken Ne(c)ks and Broken DNA: The Role of Human NEKs in the DNA Damage Response

NIMA-related kinases, or NEKs, are a family of Ser/Thr protein kinases involved in cell cycle and mitosis, centrosome disjunction, primary cilia functions, and DNA damage responses among other biological functional contexts in vertebrate cells. In human cells, there are 11 members, termed NEK1 to 11, and the research has mainly focused on exploring the more predominant roles of NEKs in mitosis regulation and cell cycle. A possible important role of NEKs in DNA damage response (DDR) first emerged for NEK1, but recent studies for most NEKs showed participation in DDR. A detailed analysis of the protein interactions, phosphorylation events, and studies of functional aspects of NEKs from the literature led us to propose a more general role of NEKs in DDR. In this review, we express that NEK1 is an activator of ataxia telangiectasia and Rad3-related (ATR), and its activation results in cell cycle arrest, guaranteeing DNA repair while activating specific repair pathways such as homology repair (HR) and DNA double-strand break (DSB) repair. For NEK2, 6, 8, 9, and 11, we found a role downstream of ATR and ataxia telangiectasia mutated (ATM) that results in cell cycle arrest, but details of possible activated repair pathways are still being investigated. NEK4 shows a connection to the regulation of the nonhomologous end-joining (NHEJ) repair of DNA DSBs, through recruitment of DNA-PK to DNA damage foci. NEK5 interacts with topoisomerase IIβ, and its knockdown results in the accumulation of damaged DNA. NEK7 has a regulatory role in the detection of oxidative damage to telomeric DNA. Finally, NEK10 has recently been shown to phosphorylate p53 at Y327, promoting cell cycle arrest after exposure to DNA damaging agents. In summary, this review highlights important discoveries of the ever-growing involvement of NEK kinases in the DDR pathways. A better understanding of these roles may open new diagnostic possibilities or pharmaceutical interventions regarding the chemo-sensitizing inhibition of NEKs in various forms of cancer and other diseases

Exacerbation of Autoimmune Neuro-Inflammation in Mice Cured from Blood-Stage <i>Plasmodium berghei</i> Infection

<div><p>The thymus plays an important role shaping the T cell repertoire in the periphery, partly, through the elimination of inflammatory auto-reactive cells. It has been shown that, during <i>Plasmodium berghei</i> infection, the thymus is rendered atrophic by the premature egress of CD4⁺CD8⁺ double-positive (DP) T cells to the periphery. To investigate whether autoimmune diseases are affected after <i>Plasmodium berghei</i> NK65 infection, we immunized C57BL/6 mice, which was previously infected with <i>P.berghei</i> NK65 and treated with chloroquine (CQ), with MOG_35–55 peptide and the clinical course of Experimental Autoimmune Encephalomyelitis (EAE) was evaluated. Our results showed that NK65+CQ+EAE mice developed a more severe disease than control EAE mice. The same pattern of disease severity was observed in MOG_35–55-immunized mice after adoptive transfer of <i>P.berghei</i>-elicited splenic DP-T cells. The higher frequency of IL-17⁺- and IFN-γ⁺-producing DP lymphocytes in the Central Nervous System of these mice suggests that immature lymphocytes contribute to disease worsening. To our knowledge, this is the first study to integrate the possible relationship between malaria and multiple sclerosis through the contribution of the thymus. Notwithstanding, further studies must be conducted to assert the relevance of malaria-induced thymic atrophy in the susceptibility and clinical course of other inflammatory autoimmune diseases.</p></div

Aggravation of EAE in mice cured from malaria correlates with increased cellular immune response towards myelin.

<p>C57BL/6 mice (n = 6 mice/group) were intraperitoneally (i.p.) infected with 1×10⁶<i>P.berghei</i>-infected Red Blood Cells and treated with chloroquine (CQ, 5 mg/Kg) for five consecutive days starting at the 10^th day after infection. Three days after the last dose of CQ, mice were immunized with 100 µg of MOG_35–55 peptide and Pertussis toxin was administrated (via i.p.) at 0 and 48h after peptide immunization for EAE induction. A) The clinical course of EAE was then monitored. Linear regression analyses are exposed in the side panels, thinner lines indicate 95% confidence interval. B) At the 10^th day after MOG-immunization, the spleens of mice were collected and dissociated. Total leukocytes (5×10⁵/well) were CFSE-stained (2,5 µM) and cultured in the presence of MOG_35–55 (10µg/mL) peptide for 96h. At the end of culture period, the cells were surface stained with anti-CD3/CD4/CD8 antibody cocktail and events were acquired in a flow cytometer. The proliferation was analyzed inside each T cell population. C) The culture supernatants were assayed for the secreted cytokines IL-10, IL-4, IL-6, IL-17, TNF-α and IFN-γ. Data was analyzed by One-Way Anova and post-tested with Bonferroni. In all analyses, *: p<0,05; ns: not significant. Representative data of three independent experiments.</p

Inflammation in the CNS of NK+CQ+EAE mice correlates with an increased production of inflammatory cytokines by DP-T cells.

<p>Groups of mice (n = 6 mice/group) subjected to infection and EAE induction. A) At the 10^th day after MOG-immunization, mice were killed and spinal cords were removed to analyze the gene expression of IL-17, IFN-γ, Foxp3 and IL-10 in the lumbar spinal cords of mice. Data was analyzed by One-Way Anova and post-tested with Bonferroni. B) The infiltrating cells of the CNS were enriched and stimulated by Phorbol Myristate Acetate and Ionomycin in the presence of Brefeldin A for 4 h. The frequency of IFN-γ- and IL-17-producing cells inside CD4⁺CD8⁺ T cell gate was analyzed. In all analyses, *: p<0,05. ns: not significant. Representative data of three independent experiments.</p

Hypothesis model for the exacerbation of autoimmune neuro-inflammation.

<p>Based in our observations, we propose a model for EAE exacerbation. Malaria infection promotes thymic atrophy and the premature egress of DP-T cells to the peripheral immune system. After an inflammatory trigger, which can be infection, genetic susceptibility or chronic inflammation, these cells proliferate and migrate to the target organ where they stimulate CNS inflammation by secreting cytokines. However, there is still much to be explored, as for example, whether these DP-T cells are able to induce leukocyte recruitment, microglia and astrocyte activation, and, Blood-Brain Barrier (BBB) destruction.</p

Central Nervous System of malaria-cured EAE mice show increased cellular infiltration of DP-T cells.

<p>C57BL/6 mice (n = 6 mice/group) were intraperitoneally (i.p.) infected with 1×10⁶<i>P.berghei</i>-infected Red Blood Cells and treated with chloroquine (CQ, 5 mg/Kg) for five consecutive days starting at the 10^th day after infection. Three days after the last dose of CQ, EAE was induced. As controls, naïve mice were treated with CQ or vehicle before EAE induction. The spinal cords of EAE-inflicted mice were collected fourteen days after MOG-immunization. Frozen thin sections (12 µm) were made and fixed in formalin. Cells were stained with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 and analyzed in epifluorescence microscope. Figures are representative of three independent experiments. Magnification: 200X.</p

Violacein treatment up-regulates the frequency of Regulatory T cells.

<p>C57BL/6 mice (n = 6 mice/group) were treated with viola (1.75, 3.5 and 7 mg/Kg) for three consecutive days. A) Kaplan-Meier curve of survival show that mice receiving the 7mg/Kg dose died after the second administration. B) After the last dose of viola, mice were killed and spleen cells were stained for flow cytometry analysis of CD4⁺CD25⁺FOXP3⁺ (Treg) cells. Results show an increase in the frequency of Treg cells after viola treatment. C) Expression of CTLA-4 was evaluated inside the Treg cell population by flow cytometry. D) The intracellular cytokine detection was performed in spleen cells from PBS- and viola-treated mice. The spleen cells were stimulated with PMA (50 ng/mL) and Ionomycin (500 ng/mL) in the presence of Brefeldin A (1 μg/mL) for 4h at 37°C. Later cells were surface stained with antibody against CD4. Following permeabilization, cells were incubated with antibody cocktail for the detection of IL-17, IL-10 and IFN-γ and preparations were acquired in Flow Cytometer equipment. Representative data from three independent experiments. All values in bar graphs are represented as means ± standard error mean. *p<0.05.</p

Violacein-elicited regulatory T cells present an enhanced suppressive activity than “naïve” Treg cells.

<p>C57BL/6 mice (n = 6 mice/group) were treated with viola (3.5mg/Kg) for three consecutive days. A) After the last dose of viola, mice were killed and spleen cells were prepared for single cell suspension. CD4⁺CD25⁺ and CD4⁺CD25^- cells were isolated using dynabeads, following manufacturer’s instructions (Life Technologies). As controls, cells were isolated from spleens from naïve mice. Treg cells were seeded in U-bottom 96-wells culture plate in increasing numbers. C57BL/6 mice (n = 3) were immunized with MOG peptide. After seven days, mice were killed and the spleens were collected and disrupted for the isolation of dendritic cells and total T cells with dynabeads. T cells were stained with CFSE (1,5 μM) and seeded to the plates containing Treg cells (5x10⁵ cells/well). Dendritic cells were isolated as well and seeded to the wells (5x10⁴ DCs/ well). As controls, encephalitogenic T cells were cultivated without Treg cells. The plates were incubated for 72h at 37°C and the suppressive activity of Treg cells was analyzed by flow cytometry. B) Treg cells were isolated from PBS- and viola-treated mice, seeded to 96-well plate and incubated for 48h at 37 ºC. The supernatants were collected and assayed for the detection of IL-10 by CBA. Representative data from two independent experiments. All values are represented as means ± standard error mean. *: p<0.05, **: p<0,01 and ***: p<0,005. Ns: not significant.</p