Cytostatic agents (e.g. paclitaxel) that disrupt microtubule dynamics are already used for chemotherapy against diverse forms of cancer [35-37]. These act in a similar way to MAP by stabilizing polymerized microtubules, blocking mitosis and ultimately leading to cell cycle arrest and apoptosis . However, in line with our observations, the nonspecific delivery of taxanes causes off-target effects, including life-threatening toxicity towards healthy cells . This was overcome by developing microtubule-blocking ADCs such as the
comprised of FOVs. All FOVs were collected and AQUA scores were generated, but each region was reviewed on a serial H&E slide to confirm that all FOVs represented infiltrating carcinoma. FOVs with normal breast ducts or ductal carcinoma in situ were excluded from the analysis. This process is illustrated in Figures 3a and 3b. Similar to the Yale University cohort, MAP-tau expression in TAX 307S remained localized to the cytoplasmic compartment within the epithelial tumor area. A total of 15,816 indivi- dual, non-overlapping FOVs were evaluated in the TAX 307S cohort. A frequency distribution summarizing the FOVs was generated for each case in the TAX 307S cohort (Figure 3c). The median score from all FOVs from each case was used to represent that case in the final subset of 108 cases. The distribution of MAP-tau expression in TAX307 for a single patient case is illu- strated in Figure 3d. The median level of normal MAP- tau expression as previously applied in the Yale Table 2 Correlation between MAP-tau expression and clinicopathologic variables in the Yale University cohort
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+TIPs: core plus end tracking proteins; AD: Alzheimer ’ s Disease; AIS: Axonal Initial segment; AMPA: α -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; A β : amyloid- β ; CNS: Central nervous system; DDX5: DEAD-box RNA helicase 5; DNA: Deoxyribonucleic acid; EBs: End binding proteins; eIF2a: Eukaryotic translation initiation factor 2A; FMRP: fragile X mental retardation protein; FTDP-17: Frontotemporal dementia with parkinsonism linked to chromosome 17; FUS: RNA-binding protein fused sarcoma; G3BP1: GTPase-activating protein-bindingprotein 1; GluN: Glutamate [NMDA] receptor subunit; H13: Minor histocompatibility antigen; HMW: High molecular weight; IMP: Insulin-like growth factor-II mRNA-binding proteins; IRS-1: Insulin receptor substrate 1; Kb: Kilo base; KO: Knockout; lncRNA: Long non-coding RNA; LTD: Long-term depression; LTP: Long-term Potentiation; MAPs: Microtubule associated proteins; MAPT: Microtubule Associated Protein Tau; miRNA: micro RNA.; mRNA: messenger RNA.; MTs: Microtubules.; NAP: Nucleossome assembly protein.; NFTs: Neurofibrillary Tangle.; NLS: Nuclear Localization Signal.; NMD: Nonsense-mediated mRNA decay.; NMDA: N-methyl-D-aspartate.; PCH: Pericentromeric heterochromatin.; PHF: Paired-helical filaments.; PIP2: Phosphatidylinositol biphosphate.; PIP3: Phosphatidylinositol triphosphate.; PNS: Peripheral Nervous System.; PSD: Post-synaptic Density.; PTEN: Phosphatase and tensin homolog.; RBPs: RNA binding protein.; RNA: Ribonucleic acid.; SGs: Stress Granules.; SN: Substantia Nigra.; TNI: Tau Nuclear Indentations.
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Amyotrophic lateral sclerosis (ALS) is the most common adult onset neurodegenerative disorder of the motor system with a lifetime risk of 1:300 and a survival of 2-5 years after diagnosis (Factor-Litvak et al., 2013). Over 50% of patients with ALS develop a cognitive (ALSci), behavioural (ALSbi) or dysexecutive syndrome consistent with that of frontotemporal dysfunction, including a frontotemporal dementia (FTD) (Ringholz et al., 2005; Strong et al., 2009). The frequent co-existence of ALS and FTD has led to the postulate that both are two states along one disease continuum (Robberecht and Philips, 2013). Importantly, patients with frontotemporal dysfunction have a reduced survival compared to other ALS cases (Elamin et al., 2011; Elamin et al., 2013; Hu et al., 2013; Olney et al., 2005). We have previously shown that ALSci is typically associated with frontotemporal atrophy with superficial linear spongiosis affecting the frontal cortex (Wilson et al., 2001), accompanied by both neuronal and glial inclusions of microtubule associated protein tau (tau protein) (Yang et al., 2003; Yang and Strong, 2012). This finding is significantly greater than observed as a function of age (Yang et al., 2005).
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16. Pentheroudakis G, Kalogeras KT, Wirtz RM, et al. Gene expression of estrogen receptor, progesterone receptor and microtubule-associated protein Tau in high-risk early breast cancer: a quest for molecular predictors of treatment benefit in the context of a Hellenic Cooperative Oncology Group trial. Breast Cancer Res Treat. 2009;116(1):131–143. 17. Gurler H, Yu Y, Choi J, Kajdacsy-Balla AA, Barbolina MV. Three- dimensional collagen type I matrix up-regulates nuclear isoforms of the microtubule associated protein tau implicated in resistance to paclitaxel therapy in ovarian carcinoma. Int J Mol Sci. 2015;16(2):3419–3433. 18. Liu YM, Chen HL, Lee HY, Liou JP. Tubulin inhibitors: a patent review.
Higher eukaryotes have developed sophisticated strategies that allow them to regulate cellular and developmental events by the selective trafficking and localization of RNA (for a review, see references 78 and 82). Several examples of inter- cellular and long-distance RNA movement have been de- scribed for higher plants, and of these, RNA viruses have become the subject of extensive investigation with regard to the molecular mechanisms that govern intercellular macromo- lecular trafficking (40). Exemplified by Tobacco mosaic virus (TMV) and other model viruses, RNA viruses facilitate the spread of their genomes by targeting plasmodesmata (Pd), membrane-lined pores that provide cytoplasmic continuity be- tween adjacent cells and tissues (40, 65). Like other plant viruses, TMV encodes a movement protein (MP) that interacts with Pd and facilitates the intercellular passage of its genome (28, 39, 64). The MP modulates the size-exclusion limit (SEL) of the Pd (107) and also binds single-stranded nucleic acids in vitro to form an unfolded, elongated ribonucleoprotein com- plex with apparent dimensions compatible with translocation through dilated Pd (22, 23, 53). The coat protein (CP) of TMV is dispensable for cell-to-cell movement (26, 96), which is con- sistent with the transport of viral RNA (vRNA) in a nonen- capsidated form (39). Since microinjected MP spreads rapidly between cells (77, 103) and plants themselves encode proteins thought to be functionally analogous to viral MPs (65, 108), the function of MP may directly reflect mechanisms of macromo- lecular Pd transport in normal plants which are exploited by viruses for the movement of their genomes. Elucidating the
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In summary, we revealed that SePTX NPs has no evident killing effect on normal cells. In cancer cells, SePTX NPs can successfully inhibit microtubule depolymerization, induce cell cycle arrest, which is related to up-regulation of microtubule associated protein p53 and cell cycle protein CyclinB1; on the other hand, SePTX NPs can successfully induce oxidative stress, cause mitochondrial dysfunction, induce mitochondrial pathway cell apoptosis, which is related to up-regulation of autophagy related protein LC3-II. In addition, SePTX NPs were able to significantly inhibit tumor growth in Lewis lung carcinoma tumor-bearing mice while reducing side effects of PTX. These results suggest that presently developed SePTX NPs have potential in the treatment of cancer.
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Subjects were excluded if they had a history of CNS opportunistic infections or non-HIV-related developmen- tal, neurological, psychiatric, or metabolic conditions that might affect CNS functioning. For inclusion, a total of 16 age-matched cases were identified with and without encephalitis or other complications. cART status was not taken into account. Overall the resulting data are consistent with other studies indicating aberrant activation of the CDK5 [60,61] and glycogen sythanse kinase (GSK) 3β [60,62-65] signaling promoting neurodegenerative changes in HAND patients. The p25/CDK5 complex associates with NFT in AD patients , and CDK5 has been shown to abnormally phosphorylate tau . These are recognized by the AT8 and PHF-1 antibodies . Both of these phosphorylation epitope-specific tau anti- bodies were elevated in patients with HIVE and in gp120 Tg mice. The patterns of AT8 and PHF-1 immunostaining in HIVE patients differed from what has been reported in AD patients. That is, in advanced AD cases the p-tau immunoreactivity is associated with dystrophic neurites, neuropil threads, and NFT [69-73]. On the other hand in the HIVE cases and in gp120 Tg mouse model there was diffuse nonfibrillar p-tau immunostaining detected in neurons and throughout the neuropil. Increased CDK5 and p35/p25 immunoreactivities were also detected throughout the neuropil of HIVE brains and gp120 Tg mice, indicating the expression of these proteins in cellular compartments that extend into the dendritic arbor and synapses. Data on whether the patients were on cART was not provided. The diffuse pattern of p-tau immunostaining was similar to what has been described in patients with preclinical AD or in the pretangle stage [70,74], suggesting that in HIVE some of the initial trig- gering events are present  which is in agreement with the previous study by Anthony et al.  and suggests HIV patients, whether on cART or not, may develop tau pathology beyond the extent seen at the age relatively young ages of the patients of these studies and that tau pathology may reach the threshold for clinical manifest- ation as the aging process continues .
Results: We investigated here the role of the microtubule (MT) cytoskeleton in mediating NC in zebrafish embryos using the MT destabilizing and hyperstabilizing drugs nocodazole and paclitaxel respectively. We found that MTs undergo major changes in organization and stability during neurulation and are required for the timely completion of NC by promoting cell elongation and polarity. We next examined the role of Microtubule-associated protein 1B (Map1b), previously shown to promote MT dynamicity in axons. map1b is expressed earlier than previously reported, in the developing neural tube and underlying mesoderm. Loss of Map1b function using morpholinos (MOs) or δ Map1b (encoding a truncated Map1b protein product) resulted in delayed NC and duplication of the neural tube, a defect associated with impaired NC. We observed a loss of stable MTs in these embryos that is likely to contribute to the NC defect. Lastly, we found that Map1b mediates cell elongation in a cell autonomous manner and polarized protrusive activity, two cell behaviors that underlie NC and are MT-dependent.
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hypothesize that a mutation in PTEN may cause visible tau aggregates in cells. To test the hypothesis, COS-7 cells stably expressing T40RW tau were transfected with pIRES- EGFP-PtenWT, pIRES-EGFP-PtenCG or pIRES-EGFP as a control, and immunostained with anti-tau and anti-tubu- lin antibody. The expression of PTENs was represented by the expression of EGFP (Fig. 4A,E,I). The tau immunoflu- orescence was shown to only partially overlap with that of microtubules (Fig. 4D,H,L), suggesting a defect in micro- tubule binding of this mutant tau. The overexpression of wild-type PTEN did not change the cellular localization of the mutant tau or the interaction between the mutant tau and microtubules (Fig. 4H). On the other hand, upon overexpression of the mutant PTEN, we observed aggre- gates of the mutant tau in the cytosol (Fig. 4J,L), although the nature of the aggregates in how they resemble the NFTs remains to be determined. In addition, the reduced immunofluoresent colocalization between the mutant tau and microtubules indicated an impaired interaction between the two. Furthermore, we observed an abnormal pattern of tau immunostaining (Fig. 4J) and less-organ- ized microtubule structures (Fig. 4K) in the mutant PTEN transfected cells compared to those in control vector and wild-type PTEN transfected cells. Given that the expres- sion of the mutant PTEN alone in the cells did not cause disorganization of the microtubules (data not shown), the observed changes in microtubules in the mutant tau transfected cells are likely due to the formation of the mutant tau aggregates.
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GMPCPP-stabilized, ATTO-565 labelled microtubule seeds. Microtubules assembly was performed at 32°C from 15 µM brain tubulin (containing 20% ATTO 565-labeled tubulin) in the presence of 100 nM EB1-GFP and 0, 0.05 µM or 1 µM CN45 peptide (GL Biochem, Shanghai, China) in BRB80 buffer supplemented with 4 mM DTT, 1% BSA, 50 mM KCl, 1 mg/ml glucose, 70 µg/ml catalase, 580 µg/ml glucose oxydase, 0.1 % methylcellulose (4,000 centipoise). Dual-color time- lapse imaging was performed on an inverted Eclipse Ti (Nikon) microscope with an Apochromat 60X1.49 N.A oil immersion objective (Nikon), equipped with an ilas2 TIRF system (Roper Scientific), a cooled charged-coupled device camera (EMCCD Evolve 512, Photometrics) and controlled by the MetaMorph 7.7.5 software (Molecular Devices). For excitation we used 491- and 561-nm lasers. Time-lapse imaging was performed at 1 frame per 2 s with an 80-ms exposure time, during 30 minutes.
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Although initially believed to be a disorder of exclusive motor dysfunction, more recent investigations have shown that approximately 50-60% of individuals with ALS possess a concurrent frontotemporal spectrum disorder (FTSD) (Elamin et al., 2011; Hudson, 1981; Montuschi et al., 2015; Oh et al., 2014). Strong and colleagues (2009, 2017) defined diagnostic criteria to accurately describe the different variants of ALS that also contain a frontotemporal syndrome, termed ALS-FTSD. Some individuals with ALS will also present with a concurrent frontotemporal dementia (ALS-FTD) meeting the Neary or Hodge’s criteria (Hodges & Miller, 2001; Neary et al., 1998). Other affected individuals may not fully meet the criteria for FTD, but will nonetheless display detectable cognitive impairment (ALSci) or behavioural impairment (ALSbi). ALSci is defined as the diagnosis of ALS with additional evidence of impaired social function, executive and/or language dysfunction. Apathy is the most common behavioural symptom identified in patients with ALSbi with some reports suggesting it can be identified in up to 70% of individuals with the disease (Lillo, Mioshi, Zoing, Kiernan, & Hodges, 2011; Witgert et al., 2010). Other symptoms associated with the ALSbi variant include disinhibition, egocentric behaviour, and changes in grooming or dietary habits. Patients may display features of both ALSci and ALSbi, with their merged diagnostic category being ALS with cognitive and behavioural impairment (ALScbi), also described as a dysexecutive syndrome. Finally, there exists a variant whereby individuals with ALS display a concurrent dementia not specified by the other mentioned criteria: ALS-Dementia. The presence of cognitive or behavioural impairment in individuals with ALS is an important feature when making predictions regarding patient prognosis in that individuals with cognitive or behavioural impairment demonstrate a significant decreased survivorship when compared to those with isolated ALS, with one study showing a decrease in survivorship by 12 months (Elamin et al., 2011). Other forms of ALS have been categorized based on their co-occurrence with other neurological conditions, including ALS with Alzheimer’s disease (ALS-AD), and ALS with CTE (ALS-CTE).
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MNV NS3 associates with stomatin and ﬂotillin, resident host proteins within cholesterol microdomains. To investigate the association of NoV NS3 with cholesterol further, we cotransfected Vero cells with cDNA plasmids encoding MNV or NV NS3 and green ﬂuorescent protein (GFP)-tagged stomatin/prohibitin/ﬂotillin/HfK/C (SPFH) pro- teins. SPFH proteins are found to be enriched in lipid microdomains within different organelles (26). Thus, we utilized GFP– erlin-1/2, GFP-prohibitin, GFP-stomatin, and GFP-ﬂotillin to detect lipid-rich microdomains in the ER, mitochondria, endosomes, and endosomes/plasma membrane, respectively. We did not observe any association be- tween MNV or NV NS3 and GFP– erlin-1/2 or GFP prohibitin (data not shown). However, we observed signiﬁcant colocalization of MNV NS3 with stomatin and ﬂotillin (Rr ⫽ 0.68 ⫾ 0.13 and Rr ⫽ 0.48 ⫾ 0.15, respectively) (Fig. 7A to F). Interestingly, MNV NS3 predominantly associated with stomatin-GFP, particularly within the vesicular struc- tures induced within the cytoplasm (Fig. 7D to F). In contrast, NV NS3 displayed less of an association with these proteins (Rr ⫽ 0.39 ⫾ 0.14 and Rr ⫽ 0.29 ⫾ 0.15 for ﬂotillin-GFP and stomatin-GFP, respectively) (Fig. 7G to I), although some colocalization between NV NS3 and ﬂotillin-GFP in the perinuclear region was also observed (Fig. 7G to I). These results suggest that MNV NS3 may have the propensity to locate to lipid-rich microdomains within the endocytic pathway and again support a difference in the localization of the MNV and NV NS3 proteins.
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polarized cells, microtubules have to be organised asymmetrically themselves. Asymmetry in microtubule distribution and stability is regulated by multiple molecular factors, most of which are microtubule-associated proteins that locally control microtubule nucleation and dynamics. At the same time, the dynamic state of microtubules is key to the regulatory mechanisms by which microtubules regulate cell polarity, modulate cell adhesion and control force-production by the actin cytoskeleton. Here, we propose that even small alterations in microtubule dynamics can influence cell migration via several different microtubule-dependent pathways. We discuss regulatory factors, potential feedback mechanisms due to functional microtubule-actin crosstalk and implications for cancer cell motility.
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Breast cancer is the most prevalent cancer in women worldwide with a high mortality rate, and the identification of new biomarkers and targets for this disease is greatly needed. Here we present evidence that microtubule-associated protein (MAP) 7 domain-containing protein 3 (Mdp3) is highly expressed in clinical samples and cell lines of breast cancer. The expression of Mdp3 cor- relates with clinicopathological parameters indicating breast cancer malignancy. In addition, Mdp3 promotes breast cancer cell proliferation and motility in vitro and stimulates breast cancer growth and metastasis in mice. Mechanistic studies reveal that γ-tubulin interacts with and recruits Mdp3 to the centrosome and that the centrosomal localization of Mdp3 is required for its activity to promote breast cancer cell proliferation and motility. These findings suggest a critical role for Mdp3 in the growth and metastasis of breast cancer and may have important implications for the management of this disease.
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has raised the possibility that neurons may be tolerant of perturbations in the overall levels of tau protein. Collec- tively, these data suggest that reductions in tau may be a viable therapeutic application. In further support of this notion, Santacruz et al. also recently demonstrated that reducing the expression of MAPT mRNA in a Tet-Off inducible tau mouse model reversed the cognitive deficits and neuronal loss in these mice without reducing the number of tau aggregates . Therefore, tau itself rather than the aggregates and tangles, may serve as a valid ther- apeutic candidate, particularly in cases caused by familial mutations of the MAPT locus. These studies demonstrate the need for compounds to facilitate the removal of either the total pool of tau or specific aberrant forms of tau (i.e. hyperphosphorylated, misfolded, etc.). Unfortunately, however, the feasibility of large scale efforts to identify modifiers of endogenous protein species has proven tech- nically challenging. We recently developed a novel tech- nique to quantitatively measure various tau species, including an assay to analyze total tau protein while simultaneously measuring GAPDH levels as an estimate of cell viability . Using this platform, we have screened a library of off-patent compounds and have identified sev- eral that are capable of reducing tau levels in brain-derived cell lines.
Since all microtubules in all classes of nutritive tubes label equally with broad range a- and p-tubulin specific antibodies, the distribution of post-translationally modified isotypes was of interest. The distribution of tyr-tubulin containing microtubules in Notonecta nutritive tube sections taken at random was significantly different from that observed with a-tubulin. In the larger functional tubes, strong staining was restricted to microtubules forming a band at the inside edge, the microtubules in the central portion of the tubes being almost totally devoid of staining (Figure 12, arrow). The smaller redundant tubes lacked staining. In contrast, the microtubules of the surrounding follicle cells and of the oocyte were brightly stained (Figure 12 and inset). When similar sections were stained with either glu- or acetylaed tubulin specific antibodies, a very different microtubule arrangement was revealed (Figures 13 and 14 respectively). Unlike tyi— microtubules glu and acetylated microtubule staining occurrecj throughout both
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34. Laws SM, Friedrich P, Diehl-Schmid J, Müller J, Eisele T, Bäuml J, Förstl H, Kurz A, Riemenschneider M: Fine mapping of the MAPT locus using quantitative trait analysis indentifies possible causal variants in Alzheimer's disease. Mol Psychiatry 2007, 12:510-517. 35. Pittman AM, Myers AJ, Abou-Sleiman P, Fung HC, Kaleem M, Mar- lowe L, Duckworth J, Leung D, Williams D, Kilford L, Thomas N, Morris CM, Dickson D, Wood NW, Hardy J, Lees AJ, de Silva R: Linkage disequilibrium fine mapping and haplotype associa- tion analysis of the tau gene in progressive supranuclear palsy and corticobasal degeneration. J Med Genet 2005, 42:837-46.
such as AD and PD, of both are prevalent with connec- tomic pathologies  thus reflect the importance of trans-synaptic organization in maintaining brain function. The seminal publications by Heiko Braak and Eva Braak described Tau pathology may initially cause neuritic dam- age in the entorhinal cortex (EC), from where it spreads to broader brain regions of the hippocampus, and pro- gressively to inferior frontal and parietal cortex [16, 17]. Modern macro-connectomic analysis supported that Tauo- pathy spreading is preferentially initiated from the densely- connected “hub” regions in the brain, therefore implicating a likelihood of disrupting the evolutionary conserved motor and cognition controls . The EC and the hippocampus are intimately connected brain areas which ensemble prop- erties of grid cells and place cells that give rise to the func- tional circuitry of map-like spatial environment that an individual can use that for spatial navigation [19, 20]. Tau pathology spreads from the EC could cause deficits in grid cell firing and disrupt the spatial cognition in mice, similar to what being seen in AD . Indeed, it is thought that pathological Tauopathy accumulation in the EC is the first synapse where tau seeds influence memory circuits in the brain, and such pathological evolvement is associated with a progressive loss of episodic memory in AD (Fig. 1) . From the view of tau structure-function relationship in the
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syndrome critical region in gene eight termed (DGCR8) (Lee et al., 2003). The process lead to the production of small hairpin structure of 70–100 nt called precursor miRNAs (pre-miRNAs). Pre-miRNAs are exported to the cytoplasm through Exportin 5 (Kim, 2004), where they are further processed by an RNase III nuclease, Dicer to produce RNA duplex (Bernstein et al., 2001; Grishok et al., 2001; Hutvagner et al., 2001). One strand is loaded on the RNA-Induced Silencing Complex (RISC) and associated with Argonaute-2 (Ago2) to interact with the target mRNA. The miRNA-RISC complex induces mRNA downregulation through two different ways: mRNA cleavage in case of perfect com- plementarity between miRNA and target mRNA or translation inhibition if there is an imperfect binding (Wahid et al., 2010) (Figure 1). In case of perfect complementarity, Ago2 is the pro- tein involved in the cleavage of the target mRNA in humans (Liu et al., 2004). However, in animals, translational repression is the most frequent way of action for miRNAs (Huntzinger and Izaurralde, 2011; Pasquinelli, 2012), although the exact process is still unknown since is not clear if the repression occur at the ini- tiation step or during the translation process (Wahid et al., 2010). Even the mechanisms for target regulation played by miRNAs are still unclear, the target mRNA could be repressed by the pro- motion of deadenylation, sequestration of miRNAs and target by stress granules and P-Bodies (Valencia-Sanchez et al., 2006), dis- ruption of translation initiation or protein degradation caused by RISC after translation (Tang et al., 2008).
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