Microtubule Dysfunction Research Articles

Context-Specific Stress Causes Compartmentalized SARM1 Activation and Local Degeneration in Cortical Neurons.

Sterile alpha and TIR motif containing 1 (SARM1) is an inducible NADase that localizes to mitochondria throughout neurons and senses metabolic changes that occur after injury. Minimal proteomic changes are observed upon either SARM1 depletion or activation, suggesting that SARM1 does not exert broad effects on neuronal protein homeostasis. However, whether SARM1 activation occurs throughout the neuron in response to injury and cell stress remains largely unknown. Using a semiautomated imaging pipeline and a custom-built deep learning scoring algorithm, we studied degeneration in both mixed-sex mouse primary cortical neurons and male human-induced pluripotent stem cell-derived cortical neurons in response to a number of different stressors. We show that SARM1 activation is differentially restricted to specific neuronal compartments depending on the stressor. Cortical neurons undergo SARM1-dependent axon degeneration after mechanical transection, and SARM1 activation is limited to the axonal compartment distal to the injury site. However, global SARM1 activation following vacor treatment causes both cell body and axon degeneration. Context-specific stressors, such as microtubule dysfunction and mitochondrial stress, induce axonal SARM1 activation leading to SARM1-dependent axon degeneration and SARM1-independent cell body death. Our data reveal that compartment-specific SARM1-mediated death signaling is dependent on the type of injury and cellular stressor.

Molecular and Cellular Involvement in CIPN.

Many anti-cancer drugs, such as taxanes, platinum compounds, vinca alkaloids, and proteasome inhibitors, can cause chemotherapy-induced peripheral neuropathy (CIPN). CIPN is a frequent and harmful side effect that affects the sensory, motor, and autonomic nerves, leading to pain, numbness, tingling, weakness, and reduced quality of life. The causes of CIPN are not fully known, but they involve direct nerve damage, oxidative stress, inflammation, DNA damage, microtubule dysfunction, and altered ion channel activity. CIPN is also affected by genetic, epigenetic, and environmental factors that modulate the risk and intensity of nerve damage. Currently, there are no effective treatments or prevention methods for CIPN, and symptom management is mostly symptomatic and palliative. Therefore, there is a high demand for better understanding of the cellular and molecular mechanisms involved in CIPN, as well as the development of new biomarkers and therapeutic targets. This review gives an overview of the current knowledge and challenges in the field of CIPN, focusing on the biological and molecular mechanisms underlying this disorder.

Ipatasertib exhibits anti‑tumorigenic effects and enhances sensitivity to paclitaxel in endometrial cancer invitro and invivo.

Endometrial cancer is the most common gynecologic cancer and one of the only cancers for which incidence and mortality is steadily increasing. Although curable with surgery in the early stages, endometrial cancer presents a significant clinical challenge in the metastatic and recurrent setting with few novel treatment strategies emerging in the past fifty years. Ipatasertib (IPAT) is an orally bioavailable pan‑AKT inhibitor, which targets all three AKT isoforms and has demonstrated anti‑tumor activity in pre‑clinical models, with clinical trials emerging for many cancer types. In the present study, the MTT assay was employed to evaluate the therapeutic efficacy of IPAT or IPAT in combination with paclitaxel (PTX) in endometrial cancer cell lines and primary cultures of endometrial cancer. The effect of IPAT and PTX on the growth of endometrial tumors was evaluated in a transgenic mouse model of endometrial cancer. Apoptosis was assessed using cleaved caspase assays and cellular stress was assessed using ROS, JC1 and tetramethylrhodamine ethyl ester assays. The protein expression levels of markers of apoptosis and cellular stress, and DNA damage were evaluated using western blotting and immunohistochemistry. IPAT significantly inhibited cell proliferation, caused cell cycle G1 phase arrest, and induced cellular stress and mitochondrial apoptosis in a dose dependent manner in human endometrial cancer cell lines. Combined treatment with low doses of IPAT and PTX led to synergistic inhibition of cell proliferation and induction of cleaved caspase 3 activity in the human endometrial cancer cell lines and the primary cultures. Furthermore, IPAT effectively reduced tumor growth, accompanied by decreased protein expression levels of Ki67 and phosphorylation of S6 in the Lkb1fl/flp53fl/fl mouse model of endometrioid endometrial cancer. The combination of IPAT and PTX resulted in increased expression of phosphorylated‑H2AX and KIF14, markers of DNA damage and microtubule dysfunction respectively, as compared with IPAT alone, PTX alone or placebo‑treated mice. The results of the present study provide a biological rationale to evaluate IPAT and the combination of IPAT and PTX in future clinical trials for endometrial cancer.

In vivo impact of tubulin polymerization promoting protein (Tppp) knockout to the airway inflammatory response

Microtubule dysfunction has been implicated as a mediator of inflammation in multiple diseases such as disorders of the cardiovascular and neurologic systems. Tubulin polymerization promoting protein (Tppp) facilitates microtubule elongation and regulates tubulin acetylation through inhibition of cytosolic deacetylase enzymes. Pathologic alterations in microtubule structure and dynamics have been described in cystic fibrosis (CF) and associated with inflammation, however the causality and mechanism remain unclear. Likewise, Tppp has been identified as a potential modifier of CF airway disease severity. Here we directly assess the impact of microtubule dysfunction on infection and inflammation by interrogating wild type and a Tppp knockout mouse model (Tppp − / −). Mice are challenged with a clinical isolate of Pseudomonas aeruginosa-laden agarose beads and assessed for bacterial clearance and inflammatory markers. Tppp − / − mouse model demonstrate impaired bacterial clearance and an elevated inflammatory response compared to control mice. These data are consistent with the hypothesis microtubule dysregulation is sufficient to lead to CF-like airway responses in mice.

Tau mutation S356T in the three repeat isoform leads to microtubule dysfunction and promotes prion-like seeded aggregation.

Tauopathies are a group of neurodegenerative diseases, which include frontotemporal dementia (FTD) and Alzheimer's disease (AD), broadly defined by the development of tau brain aggregates. Both missense and splicing tau mutations can directly cause early onset FTD. Tau protein is a microtubule-associated protein that stabilizes and regulates microtubules, but this function can be disrupted in disease states. One contributing factor is the balance of different tau isoforms, which can be categorized into either three repeat (3R) or four repeat (4R) isoforms based on the number of microtubule-binding repeats that are expressed. Imbalance of 3R and 4R isoforms in either direction can cause FTD and neurodegeneration. There is also increasing evidence that 3R tauopathies such as Pick's disease form tau aggregates predominantly comprised of 3R isoforms and these can present differently from 4R and mixed 3R/4R tauopathies. In this study, multiple mutations in 3R tau were assessed for MT binding properties and prion-like aggregation propensity. Different missense tau mutations showed varying effects on MT binding depending on molecular location and properties. Of the mutations that were surveyed, S356T tau is uniquely capable of prion-like seeded aggregation and forms extensive Thioflavin positive aggregates. This unique prion-like tau strain will be useful to model 3R tau aggregation and will contribute to the understanding of diverse presentations of different tauopathies.

Multi‐tissue profiling of RNA and DNA‐methylation signatures of progressive tau neuropathology in tau transgenic mice

AbstractBackgroundIntracellular abnormal aggregation of hyperphosphorylated tau protein is a neuropathological hallmark associated with Alzheimer’s Disease (AD). The insoluble tau aggregates called neurofibrillary tangles lead to microtubule dysfunction and neurodegeneration with AD progression. The TG4510 transgenic mice overexpressing the P301L mutant human tau protein exhibit region‐specific tau inclusions, brain atrophy, and behavioral deficits, thereby serving as a widely used animal model of tauopathy in AD. This study aims to characterize the tissue‐specific transcriptomic and epigenomic signatures of progressive tau pathology in TG4510 mice as well as investigate the correlation of brain and blood expression and epigenomic changes.MethodUsing UMI‐based bulk RNA‐Sequencing and Reduced‐representation Bisulfite Sequencing (RRBS), we profiled the gene expression and DNA‐methylation changes respectively, in the cortex, hippocampus, cerebellum and pre‐sacrifice and terminal whole blood between three age groups (2‐3 months, 6‐7 months, 8‐9 months old; N=20/tissue/group) of male and female TG4510 mice.ResultThe cortex and hippocampus show the largest transcriptional differences between mice, recapitulating phenotype differences with loss of neurotransmission and increased inflammation in these tissues. Systems‐level co‐expression analysis reveals common gene modules in immune system, behaviour and cognition and ECM/synaptic pathways between cortex/hippocampus and blood. Furthermore, the genes correlated between brain regions and blood in these mice were prioritized as potential biomarkers of tau pathology, which can be further validated in preclinical animal models and ultimately human studies.ConclusionThis study serves as one of the first evidence of multiomic molecular underpinnings in a tauopathy mouse model providing potential novel targets and biomarkers of progressive tau pathology in AD.DisclosuresAll authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

Candida albicans Reactive Oxygen Species (ROS)-Dependent Lethality and ROS-Independent Hyphal and Biofilm Inhibition by Eugenol and Citral.

Candida albicans is part of the normal human flora but is most frequently isolated as the causative opportunistic pathogen of candidiasis. Plant-based essential oils and their components have been extensively studied as antimicrobials, but their antimicrobial impacts are poorly understood. Phenylpropenoids and monoterpenes, for example, eugenol from clove and citral from lemon grass, are potent antifungals against a wide range of pathogens. We report the cellular response of C. albicans to eugenol and citral, alone and combined, using biochemical and microscopic assays. The MICs of eugenol and citral were 1,000 and 256 μg/mL, respectively, with the two exhibiting additive effects based on a fractional inhibitory concentration index of 0.83 ± 0.14. High concentrations of eugenol caused membrane damage, oxidative stress, vacuole segregation, microtubule dysfunction and cell cycle arrest at the G1/S phase, and while citral had similar impacts, they were reactive oxygen species (ROS) independent. At sublethal concentrations (1/2 to 1/4 MIC), both oils disrupted microtubules and hyphal and biofilm formation in an ROS-independent manner. While both compounds disrupt the cell membrane, eugenol had a greater impact on membrane dysfunction. This study shows that eugenol and citral can induce vacuole and microtubule dysfunction, along with the inhibition of hyphal and biofilm formation. IMPORTANCE Candida albicans is a normal resident on and in the human body that can cause relatively benign infections. However, when our immune system is severely compromised (e.g., cancer chemotherapy patients) or underdeveloped (e.g., newborns), this fungus can become a deadly pathogen, infecting the bloodstream and organs. Since there are only a few effective antifungal agents that can be used to combat fungal infections, these fungi have been exposed to them over and over again, allowing the fungi to develop resistance. Instead of developing antifungal agents that kill the fungi, some of which have undesirable side effects on the human host, researchers have proposed to target the fungal traits that make the fungus more virulent. Here, we show how two components of plant-based essential oils, eugenol and citral, are effective inhibitors of C. albicans virulence traits.

Rosemary essential oil and its components 1,8-cineole and α-pinene induce ROS-dependent lethality and ROS-independent virulence inhibition in Candida albicans.

The essential oil from Rosmarinus officinalis L., a composite mixture of plant-derived secondary metabolites, exhibits antifungal activity against virulent candidal species. Here we report the impact of rosemary oil and two of its components, the monoterpene α-pinene and the monoterpenoid 1,8-cineole, against Candida albicans, which induce ROS-dependent cell death at high concentrations and inhibit hyphal morphogenesis and biofilm formation at lower concentrations. The minimum inhibitory concentrations (100% inhibition) for both rosemary oil and 1,8-cineole were 4500 μg/ml and 3125 μg/ml for α-pinene, with the two components exhibiting partial synergy (FICI = 0.55 ± 0.07). At MIC and 1/2 MIC, rosemary oil and its components induced a generalized cell wall stress response, causing damage to cellular and organelle membranes, along with elevated chitin production and increased cell surface adhesion and elasticity, leading to complete vacuolar segregation, mitochondrial depolarization, elevated reactive oxygen species, microtubule dysfunction, and cell cycle arrest mainly at the G1/S phase, consequently triggering cell death. Interestingly, the same oils at lower fractional MIC (1/8-1/4) inhibited virulence traits, including reduction of mycelium (up to 2-fold) and biofilm (up to 4-fold) formation, through a ROS-independent mechanism.

420 Microtubule dysfunction as a mechanism of cystic fibrosis inflammation

420 Microtubule dysfunction as a mechanism of cystic fibrosis inflammation

Childhood-Onset Neurodegeneration with Cerebellar Atrophy Syndrome: Severe Neuronal Degeneration and Cardiomyopathy with Loss of Tubulin Deglutamylase Cytosolic Carboxypeptidase 1

The cytoskeleton is a dynamic filamentous network with various cellular and developmental functions. The loss of cytosolic carboxypeptidase 1 (CCP1) causes neuronal death. Childhood-onset neurodegeneration with cerebellar atrophy (CONDCA, OMIM no.: 618276) is an extremely rare disease caused by ATP/GTP binding protein 1 (AGTPBP1) gene-related CCP1 dysfunction of microtubules affecting the cerebellum, spinal motor neurons, and peripheral nerves. Also, possible problems are expected in tissues where the cytoskeleton plays a dynamic role, such as cardiomyocytes. In the present study, we report a novel homozygous missense (NM_015239: c.2447A > C, p. Gln816Pro) variant in the AGTPBP1 gene that c.2447A > C variant has never been reported in a homozygous state in the Genome Aggregation (gnomAD; v2.1.1) database, identified by whole-exome sequencing in a patient with a seizure, dystonia, dilated cardiomyopathy (DCM), and accompanying atrophy of caudate nuclei, putamen, and cerebellum. Unlike other cases in the literature, we expand the phenotype associated with AGTPBP1 variants to include dysmorphic features, idiopathic DCM which could be reversed with supportive treatments, seizure patterns, and radiological findings. These findings expanded the spectrum of the AGTPBP1 gene mutations and associated possible manifestations. Our study may help establish appropriate genetic counseling and prenatal diagnosis for undiagnosed neurodegenerative patients.

A dominant tubulin mutation causes cerebellar neurodegeneration in a genetic model of tubulinopathy.

Mutations in tubulins cause distinct neurodevelopmental and degenerative diseases termed “tubulinopathies”; however, little is known about the functional requirements of tubulins or how mutations cause cell-specific pathologies. Here, we identify a mutation in the gene Tubb4a that causes degeneration of cerebellar granule neurons and myelination defects. We show that the neural phenotypes result from a cell type–specific enrichment of a dominant mutant form of Tubb4a relative to the expression other β-tubulin isotypes. Loss of Tubb4a function does not underlie cellular pathology but is compensated by the transcriptional up-regulation of related tubulin genes in a cell type–specific manner. This work establishes that the expression of a primary tubulin mutation in mature neurons is sufficient to promote cell-autonomous cell death, consistent with a causative association of microtubule dysfunction with neurodegenerative diseases. These studies provide evidence that mutations in tubulins cause specific phenotypes based on expression ratios of tubulin isotype genes.

KANSER TEDAVİSİNDE KULLANILAN ANTİ-MİKROTÜBÜL İLAÇLARDAN VİNCA ALKALOİDLER VE PAKLİTAKSEL’İN DNA BAĞLANMA AKTİVİTELERİ

OBJECTIVE: Microtubules are an essential part of the intracellular cytoskeletal structure and possess unique polymerization dynamics that are critical for many cellular functions, including cell division. Anti-microtubule drugs that interfere with microtubule formation are important chemotherapeutic agents for the treatment of various cancer. These drugs that block mitosis seem to work by a common mechanism, which suppresses the dynamics of microtubules, slows cells, induces apoptosis and subsequently kills tumor cells. Vinca alkaloids (vinblastine, vincristine and vinorelbine) and Taxanes (paclitaxel) are two different classes of anti-microtubule drugs that cause microtubule dysfunction and inhibit cancer cell proliferation. The main activity of vinca alkaloids and taxanes result from their binding interactions with tubulin proteins. However, studies on DNA interactions of these anti-microtubule drugs are not sufficient. In this study, it was aimed to investigate the DNA binding activities of the vinca alkaloids (vinblastine, vincristine, vinorelbine) and paclitaxel.MATERIAL AND METHODS: The interactions of the drugs with DNA were analyzed by agarose gel electrophoresis assay. Three types of DNA were used in each experiment, including 100bp marker DNA, pUC19 plasmid DNA (2686 bp), and pBR322 plasmid DNA (4361 bp). After the DNAs were incubated with different concentrations of the drugs under certain conditions, agarose gel electrophoresis was performed. DNA band distributions were analyzed with a gel analysis system so that the drugs-DNA interactions could be interpreted.RESULTS: According to our results, it was found that among the vinca alkaloids, especially vinorelbine binds to DNA with higher activity than vincristine and vinblastine. The Vinca alkaloids have structural properties required for DNA binding activity and there is a similarity in their DNA binding models. However, the results showed that paclitaxel, which is from the taxane group, did not have DNA binding activity. This may be because the chemical structure of paclitaxel is not suitable for binding to DNA.CONCLUSIONS: The interaction of drugs with DNA play an important role in determining the pathways of drugs action and their ability to cause DNA damage. Consequently, the findings of our study will contribute to elucidating the effect mechanisms and the genotoxic potentials of these drugs, which are microtubule inhibitors.

Nanoscale characterization of drug-induced microtubule filament dysfunction using super-resolution microscopy

BackgroundThe integrity of microtubule filament networks is essential for the roles in diverse cellular functions, and disruption of its structure or dynamics has been explored as a therapeutic approach to tackle diseases such as cancer. Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt microtubules. However, due to associated side effects on healthy cells, there is a need to develop safer drug regimens that still retain clinical efficacy. Currently, many questions remain open regarding the extent of effects on cellular physiology of microtubule-interacting drugs at clinically relevant and low doses. Here, we use super-resolution microscopies (single-molecule localization and optical fluctuation based) to reveal the initial microtubule dysfunctions caused by nanomolar concentrations of colcemid.ResultsWe identify previously undetected microtubule (MT) damage caused by clinically relevant doses of colcemid. Short exposure to 30–80 nM colcemid results in aberrant microtubule curvature, with a trend of increased curvature associated to increased doses, and curvatures greater than 2 rad/μm, a value associated with MT breakage. Microtubule fragmentation was detected upon treatment with ≥ 100 nM colcemid. Remarkably, lower doses (< 20 nM after 5 h) led to subtle but significant microtubule architecture remodelling characterized by increased curvature and suppression of microtubule dynamics.ConclusionsOur results support the emerging hypothesis that microtubule-interacting drugs induce non-mitotic effects in cells, and establish a multi-modal imaging assay for detecting and measuring nanoscale microtubule dysfunction. The sub-diffraction visualization of these less severe precursor perturbations compared to the established antimitotic effects of microtubule-interacting drugs offers potential for improved understanding and design of anticancer agents.

Modeling a disease-correlated tubulin mutation in budding yeast reveals insight into MAP-mediated dynein function

Mutations in the genes that encode α- and β-tubulin underlie many neurological diseases, most notably malformations in cortical development. In addition to revealing the molecular basis for disease etiology, studying such mutations can provide insight into microtubule function and the role of the large family of microtubule effectors. In this study, we use budding yeast to model one such mutation—Gly436Arg in α-tubulin, which is causative of malformations in cortical development—in order to understand how it impacts microtubule function in a simple eukaryotic system. Using a combination of in vitro and in vivo methodologies, including live cell imaging and electron tomography, we find that the mutant tubulin is incorporated into microtubules, causes a shift in α-tubulin isotype usage, and dramatically enhances dynein activity, which leads to spindle-positioning defects. We find that the basis for the latter phenotype is an impaired interaction between She1—a dynein inhibitor—and the mutant microtubules. In addition to revealing the natural balance of α-tubulin isotype utilization in cells, our results provide evidence of an impaired interaction between microtubules and a dynein regulator as a consequence of a tubulin mutation and sheds light on a mechanism that may be causative of neurodevelopmental diseases.

Pathogenic MAPT mutations Q336H and Q336R have isoform-dependent differences in aggregation propensity and microtubule dysfunction.

Tauopathies are a group of heterogeneous neurodegenerative disorders characterized by brain deposition of tau inclusions. These insidious disorders include Alzheimer's disease and frontotemporal dementia, the two leading causes of dementia. Mutations in the microtubule-associated protein tau (MAPT) gene lead to familial forms of frontotemporal dementia. Previously, we used cell-based assays to screen over 20 missense tau mutations and found that decreased microtubule (MT) binding affinity was the most shared property. As a break from this trend, the MAPT mutations Q336H and Q336R are thought to promote MT assembly rather than inhibit it based on in vitro studies. Q336H and Q336R MAPT mutations also cause early onset frontotemporal dementia with Pick bodies, which are mostly composed of 3R tau isoforms. To provide further insights on the pathobiology of these mutations, we assessed Q336H and Q336R tau mutants for aggregation propensity and MT binding in cell-based assays in the context of both 0N3R and 0N4R tau isoforms. Q336R tau was prone to prion-like seeded aggregation but both Q336H and Q336R tau led to increased MT binding. Additionally, we found that different tau isoforms with these mutations heterogeneously regulate different MT subpopulations of tyrosinated and acetylated MTs, markers of newly formed MTs and stable MTs. The Q336H and Q336R tau mutations may exemplify an alternative mechanism where pathogenic tau can bind MTs with higher affinity and hyperstabilize MTs, which prevent proper MT regulation and homeostasis.

Hematological findings associated with tubulin-folding cofactors D-related encephalopathy: Expanding the phenotype.

The dysfunction of microtubules (α/β-tubulin polymers) underlies a wide range of nervous system genetic abnormalities. Defects in TBCD, a tubulin-folding cofactor, cause diseases highlighted with early-onset encephalopathy with or without neurodegeneration, intellectual disability, seizures, microcephaly and tetraparaperesis. Utilizing various molecular methods, we describe nine patients from four unrelated families with two novel exon 18 variants in TBCD exhibiting the typical neurological phenotype of the disease. Interestingly, all the investigated patients had previously unreported hematological findings in the form of neutropenia and mild degree of anemia and thrombocytopenia. In addition to delineating the neurological phenotype in several patients with TBCD variants, our study stresses on the new association of neutropenia, in particular, with the disease.

Axon Injury-Induced Autophagy Activation Is Impaired in a C. elegans Model of Tauopathy.

Autophagy is a conserved pathway that plays a key role in cell homeostasis in normal settings, as well as abnormal and stress conditions. Autophagy dysfunction is found in various neurodegenerative diseases, although it remains unclear whether autophagy impairment is a contributor or consequence of neurodegeneration. Axonal injury is an acute neuronal stress that triggers autophagic responses in an age-dependent manner. In this study, we investigate the injury-triggered autophagy response in a C. elegans model of tauopathy. We found that transgenic expression of pro-aggregant Tau, but not the anti-aggregant Tau, abolished axon injury-induced autophagy activation, resulting in a reduced axon regeneration capacity. Furthermore, axonal trafficking of autophagic vesicles were significantly reduced in the animals expressing pro-aggregant F3ΔK280 Tau, indicating that Tau aggregation impairs autophagy regulation. Importantly, the reduced number of total or trafficking autophagic vesicles in the tauopathy model was not restored by the autophagy activator rapamycin. Loss of PTL-1, the sole Tau homologue in C. elegans, also led to impaired injury-induced autophagy activation, but with an increased basal level of autophagic vesicles. Therefore, we have demonstrated that Tau aggregation as well as Tau depletion both lead to disruption of injury-induced autophagy responses, suggesting that aberrant protein aggregation or microtubule dysfunction can modulate autophagy regulation in neurons after injury.

Correction of microtubule defects within Aβ plaque-associated dystrophic axons results in lowered Aβ release and plaque deposition.

The hallmark pathologies of the Alzheimer's disease (AD) brain are amyloid beta (Aβ)-containing senile plaques and neurofibrillary tangles formed from the microtubule (MT)-binding tau protein. Tau becomes hyperphosphorylated and disengages from MTs in AD, with evidence of resulting MT structure/function defects. Brain-penetrant MT-stabilizing compounds can normalize MTs and axonal transport in mouse models with tau pathology, thereby reducing neuron loss and decreasing tau pathology. MT dysfunction is also observed in dystrophic axons adjacent to Aβ plaques, resulting in accumulation of amyloid precursor protein (APP) and BACE1 with the potential for enhanced localized Aβ generation. We have examined whether the brain-penetrant MT-stabilizing compound CNDR-51657 might decrease plaque-associated axonal dystrophy and Aβ release in 5XFAD mice that develop an abundance of Aβ plaques. Administration of CNDR-51657 to 1.5-month-old male and female 5XFAD mice for 4 or 7 weeks led to decreased soluble brain Aβ that coincided with reduced APP and BACE1 levels, resulting in decreased formation of insoluble Aβ deposits. These data suggest a vicious cycle whereby initial Aβ plaque formation causes MT disruption in nearby axons, resulting in the local accumulation of APP and BACE1 that facilitates additional Aβ generation and plaque deposition. The ability of a MT-stabilizing compound to attenuate this cycle, and also reduce deficits resulting from reduced tau binding to MTs, suggests that molecules of this type hold promise as potential AD therapeutics.

Tau Ser208 phosphorylation promotes aggregation and reveals neuropathologic diversity in Alzheimer\u2019s disease and other tauopathies

Tau protein abnormally aggregates in tauopathies, a diverse group of neurologic diseases that includes Alzheimer’s disease (AD). In early stages of disease, tau becomes hyperphosphorylated and mislocalized, which can contribute to its aggregation and toxicity. We demonstrate that tau phosphorylation at Ser208 (pSer208) promotes microtubule dysfunction and tau aggregation in cultured cells. Comparative assessment of the epitopes recognized by antibodies AT8, CP13, and 7F2 demonstrates that CP13 and 7F2 are specific for tau phosphorylation at Ser202 and Thr205, respectively, independently of the phosphorylation state of adjacent phosphorylation sites. Supporting the involvement of pSer208 in tau pathology, a novel monoclonal antibody 3G12 specific for tau phosphorylation at Ser208 revealed strong reactivity of tau inclusions in the brains of PS19 and rTg4510 transgenic mouse models of tauopathy. 3G12 also labelled neurofibrillary tangles in brains of patients with AD but revealed differential staining compared to CP13 and 7F2 for other types of tau pathologies such as in neuropil threads and neuritic plaques in AD, tufted astrocytes in progressive supranuclear palsy and astrocytic plaques in corticobasal degeneration. These results support the hypothesis that tau phosphorylation at Ser208 strongly contributes to unique types of tau aggregation and may be a reliable marker for the presence of mature neurofibrillary tangles.

Targeting microtubules sensitizes drug resistant lung cancer cells to lysosomal pathway inhibitors.

Oncogene-addicted cancers are predominantly driven by specific oncogenic pathways and display initial exquisite sensitivity to designer therapies, but eventually become refractory to treatments. Clear understanding of lung tumorigenic mechanisms is essential for improved therapies.Methods: Lysosomes were analyzed in EGFR-WT and mutant cells and corresponding patient samples using immunofluorescence or immunohistochemistry and immunoblotting. Microtubule organization and dynamics were studied using immunofluorescence analyses. Also, we have validated our findings in a transgenic mouse model that contain EGFR-TKI resistant mutations.Results: We herein describe a novel mechanism that a mutated kinase disrupts the microtubule organization and results in a defective endosomal/lysosomal pathway. This prevents the efficient degradation of phosphorylated proteins that become trapped within the endosomes and continue to signal, therefore amplifying downstream proliferative and survival pathways. Phenotypically, a distinctive subcellular appearance of LAMP1 secondary to microtubule dysfunction in cells expressing EGFR kinase mutants is seen, and this may have potential diagnostic applications for the detection of such mutants. We demonstrate that lysosomal-inhibitors re-sensitize resistant cells to EGFR tyrosine-kinase inhibitors (TKIs). Identifying the endosome-lysosome pathway and microtubule dysfunction as a mechanism of resistance allows to pharmacologically intervene on this pathway.Conclusions: We find that the combination of microtubule stabilizing agent and lysosome inhibitor could reduce the tumor progression in EGFR TKI resistant mouse models of lung cancer.