Microtubule-binding Protein Research Articles

Schizophrenia risk-gene Crmp2 deficiency causes precocious critical period plasticity and deteriorated binocular vision

Brain-specific loss of a microtubule-binding protein collapsin response mediator protein-2 (CRMP2) in the mouse recapitulates many schizophrenia-like behaviors of human patients, possibly resulting from associated developmental deficits in neuronal differentiation, path-finding, and synapse formation. However, it is still unclear how the Crmp2 loss affects neuronal circuit function and plasticity. By conducting in vivo and ex vivo electrophysiological recording in the mouse primary visual cortex (V1), we reveal that CRMP2 exerts a key regulation on the timing of postnatal critical period (CP) for experience-dependent circuit plasticity of sensory cortex. In the developing V1, the Crmp2 deficiency induces not only a delayed maturation of visual tuning functions but also a precocious CP for visual input-induced ocular dominance plasticity and its induction activity – coincident binocular inputs right after eye-opening. Mechanistically, the Crmp2 deficiency accelerates the maturation process of cortical inhibitory transmission and subsequently promotes an early emergence of balanced excitatory-inhibitory cortical circuits during the postnatal development. Moreover, the precocious CP plasticity results in deteriorated binocular depth perception in adulthood. Thus, these findings suggest that the Crmp2 deficiency dysregulates the timing of CP for experience-dependent refinement of circuit connections and further leads to impaired sensory perception in later life.

Aberrantly high activation of a FoxM1\u2013STMN1 axis contributes to progression and tumorigenesis in FoxM1-driven cancers

Fork-head box protein M1 (FoxM1) is a transcriptional factor which plays critical roles in cancer development and progression. However, the general regulatory mechanism of FoxM1 is still limited. STMN1 is a microtubule-binding protein which can inhibit the assembly of microtubule dimer or promote depolymerization of microtubules. It was reported as a major responsive factor of paclitaxel resistance for clinical chemotherapy of tumor patients. But the function of abnormally high level of STMN1 and its regulation mechanism in cancer cells remain unclear. In this study, we used public database and tissue microarrays to analyze the expression pattern of FoxM1 and STMN1 and found a strong positive correlation between FoxM1 and STMN1 in multiple types of cancer. Lentivirus-mediated FoxM1/STMN1-knockdown cell lines were established to study the function of FoxM1/STMN1 by performing cell viability assay, plate clone formation assay, soft agar assay in vitro and xenograft mouse model in vivo. Our results showed that FoxM1 promotes cell proliferation by upregulating STMN1. Further ChIP assay showed that FoxM1 upregulates STMN1 in a transcriptional level. Prognostic analysis showed that a high level of FoxM1 and STMN1 is related to poor prognosis in solid tumors. Moreover, a high co-expression of FoxM1 and STMN1 has a more significant correlation with poor prognosis. Our findings suggest that a general FoxM1-STMN1 axis contributes to cell proliferation and tumorigenesis in hepatocellular carcinoma, gastric cancer and colorectal cancer. The combination of FoxM1 and STMN1 can be a more precise biomarker for prognostic prediction.

Separable roles for RanGTP in nuclear and ciliary trafficking of a kinesin-2 subunit

Kinesin is part of the microtubule-binding motor protein superfamily, which serves important roles in cell division and intraorganellar transport. The heterotrimeric kinesin-2, consisting of the heterodimeric motor subunits, kinesin family member 3A/3B (KIF3A/3B), and kinesin-associated protein 3 (KAP3), is highly conserved across species from the unicellular eukaryote Chlamydomonas to humans. It plays diverse roles in cargo transport including anterograde (base to tip) trafficking in cilia. However, the molecular determinants mediating trafficking of heterotrimeric kinesin-2 itself are poorly understood. It has been previously suggested that ciliary transport is analogous to nuclear transport mechanisms. Using Chlamydomonas and human telomerase reverse transcriptase-retinal pigment epithelial cell line, we show that RanGTP, a small GTPase that dictates nuclear transport, regulates ciliary trafficking of KAP3, a key component for functional kinesin-2. We found that the armadillo-repeat region 6 to 9 (ARM6–9) of KAP3, required for its nuclear translocation, is also necessary and sufficient for its targeting to the ciliary base. Given that KAP3 is essential for cilium formation and there are the emerging roles for RanGTP/importin β in ciliary protein targeting, we further investigated the effect of RanGTP in cilium formation and maintenance. We found that precise control of RanGTP levels, revealed by different Ran mutants, is crucial for cilium formation and maintenance. Most importantly, we were able to provide orthogonal support in an algal model system that segregates RanGTP regulation of ciliary protein trafficking from its nuclear roles. Our work provides important support for the model that nuclear import mechanisms have been co-opted for independent roles in ciliary import.

Recapitulation of Endogenous 4R Tau Expression and Formation of Insoluble Tau in Directly Reprogrammed Human Neurons

Tau is a microtubule-binding protein expressed in neurons and the equal ratio between 4-repeat (4R) and 3-repeat (3R) isoforms are maintained in normal adult brain function. Dysregulation of 3R;4R ratio causes tauopathy and human neurons that recapitulate tau isoforms in health and disease will provide a platform for elucidating pathogenic processes involving tau pathology. We carried out extensive characterizations of tau isoforms expressed in human neurons derived by microRNA-induced neuronal reprogramming of adult fibroblasts. Transcript and protein analyses showed miR-neurons expressed all six isoforms with the 3R:4R isoform ratio equivalent to that detected in human adult brains. Also, miR-neurons derived from familial tauopathy patients with a 3R:4R ratio altering mutation showed increased 4R tau and the formation of insoluble tau with seeding activity. Our results collectively demonstrate the utility of miRNA-induced neuronal reprogramming to recapitulate endogenous tau regulation comparable to the adult brain in health and disease.

Tau and DNA Damage in Neurodegeneration.

Neurodegenerative disorders are a family of incurable conditions. Among them, Alzheimer’s disease and tauopathies are the most common. Pathological features of these two disorders are synaptic loss, neuronal cell death and increased DNA damage. A key pathological protein for the onset and progression of the conditions is the protein tau, a microtubule-binding protein highly expressed in neurons and encoded by the MAPT (microtubule-associated protein tau) gene. Tau is predominantly a cytosolic protein that interacts with numerous other proteins and molecules. Recent findings, however, have highlighted new and unexpected roles for tau in the nucleus of neuronal cells. This review summarizes the functions of tau in the metabolism of DNA, describing them in the context of the disorders.

GSK3α, not GSK3β, drives hippocampal NMDAR-dependent LTD via tau-mediated spine anchoring.

Glycogen synthase kinase-3 (GSK3) is an important signalling protein in the brain and modulates different forms of synaptic plasticity. Neuronal functions of GSK3 are typically attributed to one of its two isoforms, GSK3β, simply because of its prevalent expression in the brain. Consequently, the importance of isoform-specific functions of GSK3 in synaptic plasticity has not been fully explored. We now directly address this question for NMDA receptor-dependent long-term depression (LTD) in the hippocampus. Here, we specifically target the GSK3 isoforms with shRNA knock-down in mouse hippocampus and with novel isoform-selective drugs to dissect their roles in LTD. Using electrophysiological and live imaging approaches, we find that GSK3α, but not GSK3β, is required for LTD. The specific engagement of GSK3α occurs via its transient anchoring in dendritic spines during LTD induction. We find that the major GSK3 substrate, the microtubule-binding protein tau, is required for this spine anchoring of GSK3α and mediates GSK3α-induced LTD. These results link GSK3α and tau in a common mechanism for synaptic depression and rule out a major role for GSK3β in this process.

NUSAP1 Promotes Cell Proliferation Via the DNA Damage Response Pathway in Diffuse Large B-Cell Lymphoma

Introduction: Nucleolar spindle-associated protein 1 (NUSAP1), a microtubule binding protein with molecular weight of 55KD, plays an important role to ensure the normal regulation of cell cycle in chromosome separation, spindle assembly and DNA repair. NUSAP1 has been shown to be highly expressed in a variety of tumors, involved in tumor occurrence, invasion, migration, and drug resistance. Moreover, it is associated with poor prognosis. Whereas, no research has been reported regarding the role of NUSAP1 in diffuse large B-cell lymphoma (DLBCL). Methods: Peripheral blood samples from de novo DLBCL patients and healthy volunteers were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong University (SPHASU). Microarray datasets GSE83632 and GSE32918 were obtained from Gene Expression Omnibus. Kaplan-Meier survival curves with log-rank test of overall survival (OS) were analyzed. Immunohistochemistry staining (IHC) was performed to assess NUSAP1 expression in specimens. Expression levels of NUSAP1 mRNA and protein were detected by quantitative RT-PCR and western blotting. The DLBCL cells were transfected by lentiviral shRNA and vectors to stably silence and up-regulate NUSAP1. Effects of doxorubicin on cell viabilities were assessed by cell counting kit-8. Besides, apoptosis and cell cycle were respectively detected by annexin V-PE/7AAD and PI/RNase staining via flow cytometry. Invasion ability was analyzed by transwell assay. ShNUSAP1 cells and Scramble cells were subcutaneously injected to SCID-Beige mice to establish xenograft models. Animal experiments were performed in accordance with the principles of the Institutional Animal Care. Results: According to clinical specimens and bioinformatics analysis, the expression level of NUSAP1 gene in samples of DLBCL patients was significantly increased than that of healthy donors (P<0.05) (Figure A, B). Besides, patients with high expression of NUSAP1 was related to shorter overall survival (HR=0.456, P<0.01), indicating poor prognosis (Figure C). Stronger positivity of NUSAP1 was markedly observed in DLBCL lymph nodes than reactive hyperplasia group, which was associated with Ann Arbor stage of DLBCL patients (Figure D). Compared with normal B cells, both the mRNA and protein level of NUSAP1 were up-regulated in DLBCL cell lines (Figure E, F). After transfected with lentivirus, the proliferation rate of NUSAP1 knockdown group was lower than that of the control group, while the overexpression group was faster than control (Figure G). Through flow cytometry, silencing of NUSAP1 led to increased apoptotic rates of LY1 and LY3 cells (Figure H). Along with the recovery of NUSAP1 expression level, apoptosis rates were improved again. With the addition of doxorubicin at 100nM, interference of NUSAP1 could increase the sensitivity of cells to doxorubicin (Figure I). NUSAP1 intervention induced obvious cell cycle arrest in G1 phase of LY1 and LY3 cell lines, with concomitant reduction of cell proportion in S phase (Figure J). Moreover, known as EMT biomarkers, the expression of ZEB1 and VIMENTIN were decreased with reduction of NUSAP1. Likewise, we also investigated NUSAP1 overexpression has promoted EMT-like process of LY1 and LY3 cells (Figure K). Surppression of NUSAP1 inhibited the action of DNA damage repair pathway (Figure L). Moreover, we also found that NUSAP1 knockdown induced striking reduction in invasion cells (Figure M). We found that tumor growth was significantly suppressed by inhibiting NUSAP1 in xenograft models (Figure N, O). IHC for NUSAP1 and Ki67 on tumor sections showed that tumors derived from shNUSAP1 cells exhibited significantly lower levels of Ki67 compared to control group (Figure P). Conclusions: This study first identified that the high expression of NUSAP1 in DLBCL patients is associated with poor prognosis through database analysis and in vitro experiments. Interference of NUSAP1 expression led to a slower DLBCL cell proliferation and a higher apoptosis rate, meanwhile induced the G1 phase arrest and promoted EMT-like process. Collectively, our study demonstrated that NUSAP1 plays a role in promoting tumor growth both in vivo and vitro through DNA damage response pathway, which providing a new direction for prognosis assessment and targeted therapy of DLBCL. Figure Disclosures No relevant conflicts of interest to declare.

NUSAP1 Enhances Drug Resistance By Binding to RAD51 through DNA Damage Repair Pathway in Chronic Lymphocytic Leukemia

Introduction: Enhanced DNA damage repair effect is an important mechanism for drug-resistance in chronic lymphocytic leukemia (CLL). Moreover, the ability of cancer cells to repair under radiation or chemotherapy drug induced DNA damage also serves as one of the mechanisms for therapy resistance. It is reported that nucleolar and spindle associated protein 1 (NUSAP1), a microtubule binding protein, has been involved in DNA damage repair process and plays important roles in the development, progression, and metastasis in several types of cancer. However, its role and mechanism in the development of CLL are still unclear. Methods: Expression levels of NUSAP1 mRNA and protein in CLL cell lines and patient specimens were detected by qRT-PCR and Western blot, and Kaplan-Meier survival curve and overall survival were analyzed by log-rank test. Peripheral blood samples from de novo CLL patients and healthy volunteers were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong University (SPHASU). Microarray datasets GSE22762 were obtained from Gene Expression Omnibus. With altering NUSAP1 expression by lentivirus-transfected cells in vitro, the effects of NUSAP1 on cell proliferation, apoptosis and cycle were detected by CCK8, Annexin V-PE /7AAD staining and PI/RNase staining respectively. Bioinformatics analysis, luciferase reporter analysis, immunoprecipitation and were applied to discern and examine the relationship between NUSAP1 and its potential targets. Results: According to clinical specimens and bioinformatics analysis, the expression level of NUSAP1 gene in samples of CLL patients was significantly increased than that of healthy donors (P<0.05) (Figure A). Besides, the results indicated that the OS of patients with highly expressed NUSAP1 was significantly worse than in patients with low expression with the statistical analysis database GSE22762. mRNA and protein expression levels of NUSAP1 were significantly higher in CLL cell lines than in PBMCs from healthy donors (Figure C). Our findings indicated that NUSAP1 knockdown notably inhibited cell proliferation when compared with the Scramble group (Figure D). Moreover, the amounts of DNA fragmentation of the apoptotic cells were remarkably increased by NUSAP1 shRNA in MEC-1 and EHEB cells when compared with the Scramble group (Figure E). In addition, after knocking down NUSAP1, MEC-1 and EHEB cells were blocked in G0/G1 phase (Figure F). Moreover, addition to fludarabine or ibrutinib with shNUSAP1 group showed enhanced cytotoxicity in CLL cells (Figure G). The differential genes were analyzed via RNA-seq between Scramble and ShNUSAP1 group. Intriguingly, annotations of gene ontology (GO) analysis indicated that NUSAP1 was closely related to biological processes including cell cycle and response to drug. Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that NUSAP1 were enriched in pathways in cancer, DNA replication and cell cycle. Gene set enrichment analysis (GSEA) implicated that NUSAP1 was functionally enriched in DNA replication, cell cycle and proteasome (Figure H). Immunofluorescence showed that NUSAP1 was mainly distributed in the cell nucleus, and the expression level of RAD51 was positively correlated with the change of NUSAP1 expression (Figure I). Surppression of NUSAP1 inhibited the action of proteins in DNA damage repair pathway (Figure J). Through COIP, NUSAP1 was identified to bind with RAD51 and play an important role in DNA damage repair pathway (Figure K). Hence, NUSAP1 participates in the DNA damage repair process and enhances the drug resistance in CLL. Conclusions: This study first demonstrated that the high expression of NUSAP1 in CLL patients is associated with poor prognosis through database analysis and experiments in vitro. Interference of NUSAP1 expression led to a slower CLL cell proliferation and a higher apoptosis rate, meanwhile induced the G1 phase arrest. Collectively, our findings demonstrated that NUSAP1 contributes to DNA damage repairing by binding to RAD51 and enhances drug resistance in CLL. Therefore, NUSAP1 is expected to be a potential target for the treatment of CLL with drug-resistance. Figure 1 Disclosures No relevant conflicts of interest to declare.

Characterization of Synergistic Selinexor Combinations of Dexamethasone, Pomalidomide, Elotuzumab and Daratumumab in Primary MM Samples Ex Vivo

Introduction. Multiple myeloma (MM) is an incurable plasma cell malignancy with a growing list of anti-MM therapeutics. However, the development of predictive biomarkers has yet to be achieved for nearly all MM therapeutics. Selinexor (SELI), a nuclear export inhibitor targeting exportin 1 (XPO1), has been approved with dexamethasone (DEX) in penta-refractory MM. Clinical studies investigating promising SELI- 3 drug combinations are ongoing. Here, we have investigated potential synergistic combinations of SELI and anti-MM agents in terms of ex vivo sensitivity, as well as paired RNAseq and WES to identify companion biomarkers. Methods. MM cells isolated from fresh bone marrow aspirates were tested for drug sensitivity in an organotypic ex vivo drug sensitivity assay, consisting of co-culture with stroma, collagen matrix and patient-derived serum. Single agents were tested at 5 concentrations, while two-drug combinations were tested at fixed ratio of concentrations. LD50 and area under the curve (AUC) were assessed during 96h-exposure as metrics for drug resistance. Drug synergy was calculated as a modified BLISS model. Matching aliquots of MM cells had RNAseq and WES performed through ORIEN/AVATAR project. Geneset enrichment analysis (GSEA) was conducted using both AUC and LD50 as phenotypes for single agents and combinations. Both curated pathways (KEGG and cancer hallmarks) and unsupervised gene clustering were used as genesets. Student t-tests with multiple test correction were used to identify non-synonymous mutations in protein coding genes associated with single agent or combination AUC. Results. For this analysis, a cohort of specimens from 103 patients (48% female, 4% Hispanic, 11% African American) was tested with SELI and/or DEX. with a median of 2 lines of therapy (0-12). A smaller cohort of 37 have been examined with SELI, pomalidomide (POM), elotuzumab (ELO) and daratumumab (DARA). Within this cohort we observed synergy between SELI and DEX, POM and ELO as shown in Figure 1. The volcano plot illustrates the number of samples, maximum drug concentration, as well as magnitude (x- axis) and significance (y- axis) of synergy. Although the SELI-DARA combination trended toward synergy, statistical significance was not achieved. To identify molecular mechanisms and biomarkers associated with sensitivity to SELI and SELI- combinations, we investigated paired RNAseq and WES with ex vivo sensitivity. Initially, we conducted GSEA on two cohorts of primary MM samples tested with SELI alone at 5µM (n=53) and 10µM (n=50). Cell adhesion (KEGG CAMS), inflammatory cytokines (KEGG ASTHMA), and epithelial mesenchymal transition (HALLMARK EMT) were associated with resistance in both cohorts, while the HALLMARK MYC TARGETS was associated with sensitivity (FWER p<0.05). Mutational analysis identified 46 gene mutations associated with SELI resistance and 100 associated with sensitivity at 5µM, and 87 and 27 mutations associated with SELI resistance and sensitivity, respectively, at 10µM. Two gene mutations were identified in both cohorts: BCL7A, involved in chromatin remodeling, was associated with sensitivity and CEP290, a microtubule binding protein, associated with resistance (p<0.05). Analysis of both gene sequences (NetNES 1.1) identified nuclear export signal (NES) residues suggesting these may be XPO1 cargo. Additionally, translocation t(11;14) was associated with SELI resistance in the 5µM cohort (p=0.037). The completed set of 50 specimens ex vivo, RNAseq and WES analysis will be mature and updated for the potential presentation at ASH. Conclusions. We observed ex vivo synergy between SELI and DEX, POM and ELO. Molecular analysis of matched ex vivo drug sensitivity, transcriptome and mutational profile identified environment-mediated drug resistance pathways positively correlated with SELI single agent resistance, as well as MYC regulated genes associated with ex vivo sensitivity. We also identified a list of mutations associated with SELI drug resistance and sensitivity, with special emphasis on two novel NES-containing genes, CEP290 and BCL7A. The next step of this project is to analyze transcriptional and mutational patterns associated with ex vivo synergy in the combinations here described, as putative biomarkers for future clinical investigation. Disclosures Shain: Amgen: Speakers Bureau; Adaptive: Consultancy, Honoraria; Karyopharm: Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GlaxoSmithKline: Speakers Bureau; Janssen: Honoraria, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; AbbVie: Research Funding. Kulkarni:M2GEN: Current Employment. Zhang:M2GEN: Current Employment. Hampton:M2GEN: Current Employment. Argueta:Karyopharm: Current Employment. Landesman:Karyopharm Therapeutics Inc: Current Employment, Current equity holder in publicly-traded company. Siqueira Silva:AbbVie: Research Funding; NIH/NCI: Research Funding; Karyopharm: Research Funding.

Immune Profiling of Gliomas Reveals a Connection with IDH1/2 Mutations, Tau Function and the Vascular Phenotype.

Simple SummaryIn the present work we have confirmed that gliomas with isocitrate dehydrogenase 1/2 mutations are “cold” tumors, whereas the immune content of their wild-type counterparts is more heterogeneous. A large subgroup of wild-type glioblastomas is characterized by an important immune component, particularly enriched in myeloid cells, and an elevated expression of the ligand of programmed death ligand 1 (PD-L1) in the immune compartment. The rest contain few lymphocytes and myeloid cells. Notably, we have observed a direct correlation between the immune content and the presence of vascular alterations, as well as with the reduced expression of Tau, a microtubule-binding protein that we described as a negative regulator of angiogenesis. Using syngeneic mouse models, we show that overexpression of Tau reduces the immune content, delaying tumor growth.Background: Gliomas remain refractory to all attempted treatments, including those using immune checkpoint inhibitors. The characterization of the tumor (immune) microenvironment has been recognized as an important challenge to explain this lack of response and to improve the therapy of glial tumors. Methods: We designed a prospective analysis of the immune cells of gliomas by flow cytometry. Tumors with or without isocitrate dehydrogenase 1/2 (IDH1/2) mutations were included in the study. The genetic profile and the presence of different molecular and cellular features of the gliomas were analyzed in parallel. The findings were validated in syngeneic mouse models. Results: We observed that few immune cells infiltrate mutant IDH1/2 gliomas whereas the immune content of IDH1/2 wild-type tumors was more heterogeneous. Some of them contained an important immune infiltrate, particularly enriched in myeloid cells with immunosuppressive features, but others were more similar to mutant IDH1/2 gliomas, with few immune cells and a less immunosuppressive profile. Notably, we observed a direct correlation between the percentage of leukocytes and the presence of vascular alterations, which were associated with a reduced expression of Tau, a microtubule-binding protein that controls the formation of tumor vessels in gliomas. Furthermore, overexpression of Tau was able to reduce the immune content in orthotopic allografts of GL261 cells, delaying tumor growth. Conclusions: We have confirmed the reduced infiltration of immune cells in IDH1/2 mutant gliomas. By contrast, in IDH1/2 wild-type gliomas, we have found a direct correlation between the presence of vascular alterations and the entrance of leukocytes into the tumors. Interestingly, high levels of Tau inversely correlated with the vascular and the immune content of gliomas. Altogether, our results could be exploited for the design of more successful clinical trials with immunomodulatory molecules.

Proximity-dependent Proteomics Reveals Extensive Interactions of Protocadherin-19 with Regulators of Rho GTPases and the Microtubule Cytoskeleton

Protocadherin-19 belongs to the cadherin family of cell surface receptors and has been shown to play essential roles in the development of the vertebrate nervous system. Mutations in human Protocadherin-19 (PCDH19) lead to PCDH19 Female-limited epilepsy (PCDH19 FLE) in humans, characterized by the early onset of epileptic seizures in children and a range of cognitive and behavioral problems in adults. Despite being considered the second most prevalent gene in epilepsy, very little is known about the intercellular pathways in which it participates. In order to characterize the protein complexes within which Pcdh19 functions, we generated Pcdh19-BioID fusion proteins and utilized proximity-dependent biotinylation to identify neighboring proteins. Proteomic identification and analysis revealed that the Pcdh19 interactome is enriched in proteins that regulate Rho family GTPases, microtubule binding proteins and proteins that regulate cell divisions. We cloned the centrosomal protein Nedd1 and the RacGEF Dock7 and verified their interactions with Pcdh19 in vitro. Our findings provide the first comprehensive insights into the interactome of Pcdh19, and provide a platform for future investigations into the cellular and molecular biology of this protein critical to the proper development of the nervous system.

Nuclear Transport Deficits in Tau-Related Neurodegenerative Diseases.

Tau is a cytosolic microtubule binding protein that is highly abundant in the axons of the central nervous system. However, alternative functions of tau also in other cellular compartments are suggested, for example, in the nucleus, where interactions of tau with specific nuclear entities such as DNA, the nucleolus, and the nuclear envelope have been reported. We would like to review the current knowledge about tau–nucleus interactions and lay out possible neurotoxic mechanisms that are based on the (pathological) interactions of tau with the nucleus.

RPS23RG1 modulates tau phosphorylation and axon outgrowth through regulating p35 proteasomal degradation.

Tauopathies are a group of neurodegenerative diseases characterized by hyperphosphorylation of the microtubule-binding protein, tau, and typically feature axon impairment and synaptic dysfunction. Cyclin-dependent kinase5 (Cdk5) is a major tau kinase and its activity requires p35 or p25 regulatory subunits. P35 is subjected to rapid proteasomal degradation in its membrane-bound form and is cleaved by calpain under stress to a stable p25 form, leading to aberrant Cdk5 activation and tau hyperphosphorylation. The type Ib transmembrane protein RPS23RG1 has been implicated in Alzheimer's disease (AD). However, physiological and pathological roles for RPS23RG1 in AD and other tauopathies are largely unclear. Herein, we observed retarded axon outgrowth, elevated p35 and p25 protein levels, and increased tau phosphorylation at major Cdk5 phosphorylation sites in Rps23rg1 knockout (KO) mice. Both downregulation of p35 and the Cdk5 inhibitor roscovitine attenuated tau hyperphosphorylation and axon outgrowth impairment in Rps23rg1 KO neurons. Interestingly, interactions between the RPS23RG1 carboxyl-terminus and p35 amino-terminus promoted p35 membrane distribution and proteasomal degradation. Moreover, P301L tau transgenic (Tg) mice showed increased tau hyperphosphorylation with reduced RPS23RG1 levels and impaired axon outgrowth. Overexpression of RPS23RG1 markedly attenuated tau hyperphosphorylation and axon outgrowth defects in P301L tau Tg neurons. Our results demonstrate the involvement of RPS23RG1 in tauopathy disorders, and implicate a role for RPS23RG1 in inhibiting tau hyperphosphorylation through homeostatic p35 degradation and suppression of Cdk5 activation. Reduced RPS23RG1 levels in tauopathy trigger aberrant Cdk5-p35 activation, consequent tau hyperphosphorylation, and axon outgrowth impairment, suggesting that RPS23RG1 may be a potential therapeutic target in tauopathy disorders.

Knockin' on heaven's door: Molecular mechanisms of neuronal tau uptake.

Since aggregates of the microtubule‐binding protein tau were found to be the main component of neurofibrillary tangles more than 30 years ago, their contribution to neurodegeneration in Alzheimer's disease (AD) and tauopathies has become well established. Recent work shows that both tau load and its distribution in the brain of AD patients correlate with cognitive decline more closely compared to amyloid plaque deposition. In addition, the amyloid cascade hypothesis has been recently challenged because of disappointing results of clinical trials designed to treat AD by reducing beta‐amyloid levels, thus fuelling a renewed interest in tau. There is now robust evidence to indicate that tau pathology can spread within the central nervous system via a prion‐like mechanism following a stereotypical pattern, which can be explained by the trans‐synaptic inter‐neuronal transfer of pathological tau. In the receiving neuron, tau has been shown to take multiple routes of internalisation, which are partially dependent on its conformation and aggregation status. Here, we review the emerging mechanisms proposed for the uptake of extracellular tau in neurons and the requirements for the propagation of its pathological conformers, addressing how they gain access to physiological tau monomers in the cytosol. Furthermore, we highlight some of the key mechanistic gaps of the field, which urgently need to be addressed to expand our understanding of tau propagation and lead to the identification of new therapeutic strategies for tauopathies.

Transcriptome-wide effect of Salix SmSPR1 in etiolated seedling of Arabidopsis

Microtubules and their regulatory proteins are involved in the regulation of plant cell morphology. SPIRAL1 (SPR1), a plant-specific microtubule-binding protein, is critical in regulating the anisotropic growth of plant cells. Our previous study showed that overexpressed Salix SmSPR1 genes in Arabidopsis thaliana caused right-handed spiral elongation in etiolated seedlings, but there were no morphological differences between wild-type and transgenic seedlings under varied light conditions. We then studied the transcriptional regulation patterns in transgenic plants engineered with the SmSPR1 gene. Transcriptomic results showed that a large number of differentially expressed genes were involved in plant light signal reception, chlorophyll synthesis and photosystem structure. Eleven gene families with 42 photosynthesis-related genes and 6 light-responsive genes were involved in regulation of cell morphology. Our results showed that these genes in the SmSPR1-ox line were particularly down-regulated under dark conditions. In addition, 33 TFs showed differences between SmSPR1-ox and wild-type lines. Taken together, the transcriptome analysis provides new insight into investigating the molecular mechanisms of light-induced cell morphological changes mediated by the microtubule binding protein SPR1.

1H, 13C and 15N resonance assignments for the microtubule-binding domain of the kinetoplastid kinetochore protein KKT4 from Trypanosoma brucei

KKT4 is a kinetoplastid-specific microtubule-binding kinetochore protein that lacks significant similarity to any known kinetochore or microtubule-binding proteins. Here we present the 1H, 13C and 15N resonance assignments for several fragments from the microtubule-binding domain of KKT4 (KKT4115–343) from Trypanosoma brucei. These assignments provide the starting point for detailed investigations of the structure, dynamics and interactions of the microtubule-binding region of KKT4.

The protein-surfactant stoichiometry governs the conformational switching and amyloid nucleation kinetics of tau K18.

Amyloids are pathological hallmarks of a number of debilitating neurodegenerative diseases. Understanding the molecular mechanism of protein amyloid assembly with an emphasis on structural characterization of early, key prefibrillar species is important for targeted drug design and clinical interventions. Tau is an intrinsically disordered, microtubule-binding protein which is also implicated in various neurodegenerative disorders such as frontotemporal dementia, Down's syndrome, Alzheimer's disease, etc. Earlier reports have demonstrated that tau aggregation in vitro is triggered by anionic inducers, presumably due to charge compensation which facilitates intermolecular association between the tau polypeptide chains. However, the molecular mechanism of tau amyloid aggregation, involving the structural characterization of amyloidogenic intermediates formed especially during early key steps, remains elusive. In this work, we have employed a spectroscopic toolbox to elucidate the mechanism of anionic surfactant-induced disorder-to-order amyloid transition of a tau segment. This study revealed that the amyloid assembly is mediated via binding-induced conformational switching into an early partially helical amyloid-competent intermediate. Additionally, protein and inducer concentration-dependent studies indicated that at the higher protein and/or inducer concentrations, competing off-pathway intermediates dampen the amyloid assembly which implies that the stoichiometry of protein and inducer plays a key regulatory role in the amyloid nucleation and fibril elongation kinetics.

TANGLED1 mediates microtubule interactions that may promote division plane positioning in maize.

The microtubule cytoskeleton serves as a dynamic structural framework for mitosis in eukaryotic cells. TANGLED1 (TAN1) is a microtubule-binding protein that localizes to the division site and mitotic microtubules and plays a critical role in division plane orientation in plants. Here, in vitro experiments demonstrate that TAN1 directly binds microtubules, mediating microtubule zippering or end-on microtubule interactions, depending on their contact angle. Maize tan1 mutant cells improperly position the preprophase band (PPB), which predicts the future division site. However, cell shape-based modeling indicates that PPB positioning defects are likely a consequence of abnormal cell shapes and not due to TAN1 absence. In telophase, colocalization of growing microtubules ends from the phragmoplast with TAN1 at the division site suggests that TAN1 interacts with microtubule tips end-on. Together, our results suggest that TAN1 contributes to microtubule organization to ensure proper division plane orientation.

Novel nephronophthisis-associated variants reveal functional importance of MAPKBP1 dimerization for centriolar recruitment

Biallelic mutations in MAPKBP1 were recently associated with late-onset cilia-independent nephronophthisis. MAPKBP1 was found at mitotic spindle poles but could not be detected at primary cilia or centrosomes. Here, by identification and characterization of novel MAPKBP1 variants, we aimed at further investigating its role in health and disease. Genetic analysis was done by exome sequencing, homozygosity mapping, and a targeted kidney gene panel while coimmunoprecipitation was used to explore wild-type and mutant protein-protein interactions. Expression of MAPKBP1 in non-ciliated HeLa and ciliated inner medullary collecting duct cells enabled co-localization studies by fluorescence microscopy. By next generation sequencing, we identified two novel homozygous MAPKBP1 splice-site variants in patients with nephronophthisis-related chronic kidney disease. Splice-site analyses revealed truncation of C-terminal coiled-coil domains and patient-derived deletion constructs lost their ability to homodimerize and heterodimerize with paralogous WDR62. While wild-type MAPKBP1 exhibited centrosomal, basal body, and microtubule association, mutant proteins lost the latter and showed reduced recruitment to cell cycle dependent centriolar structures. Wild-type and mutant proteins had no reciprocal influence upon co-expression excluding dominant negative effects. Thus, MAPKBP1 appears to be a novel microtubule-binding protein with cell cycle dependent centriolar localization. Truncation of its coiled-coil domain is enough to abrogate its dimerization and results in severely disturbed intracellular localizations. Delineating the impact of impaired dimerization on cell cycle regulation and intracellular kidney signaling may provide new insights into common mechanisms of kidney degeneration. Thus, due to milder clinical presentation, MAPKBP1-associated nephronophthisis should be considered in adult patients with otherwise unexplained chronic kidney disease.

HIV influences microtubule associated protein-2: potential marker of HIV-associated neurocognitive disorders.

Postmortem brains of patients diagnosed with HIV-1-associated neurocognitive disorders (HAND) exhibit loss of dendrites. However, the mechanisms by which synapses are damaged are not fully understood. Dendrite length and remodeling occurs via microtubules, the dynamics of which are regulated by microtubule-binding proteins, including microtubule-associated protein 2 (MAP2). The HIV protein gp120 is neurotoxic and interferes with neuronal microtubules. We measured MAP2 concentrations in human cerebrospinal fluid (CSF) and MAP2 immunoreactivity in rat cortical neurons exposed to HIV and gp120. First, we examined whether HIV affects MAP2 levels by analyzing the CSF of 27 persons living with HIV (PLH) whose neurocognitive performance had been characterized. We then used rat cortical neurons to study the mechanisms of HIV-mediated dendritic loss. PLH who had HAND had greater MAP2 concentrations within the CSF than cognitive normal PLH. In cortical neurons, the deleterious effect of HIV on MAP2-positive dendrites occurred through a gp120-mediated mechanism. The neurotoxic effect of HIV was blocked by a CCR5 antagonist and prevented by Helix-A, a peptide that displaces gp120 from binding to microtubules, conjugated to a nanolipoprotein particle delivery platform. Our findings support that HIV at least partially effects its neurotoxicity via neuronal cytoskeleton modifications and provide evidence of a new therapeutic compound that could be used to prevent the HIV-associated neuropathology.