Brain Microtubules Research Articles

Could there be an experimental way to link consciousness and quantum computations of brain microtubules?

Could there be an experimental way to link consciousness and quantum computations of brain microtubules?

Quantitative Microtubule Fractionation Technique to Separate Stable Microtubules, Labile Microtubules, and Free Tubulin in Mouse Tissues.

Microtubules, composed of α/β-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit dynamic instability as tubulin heterodimer subunits undergo repetitive polymerization and depolymerization. Precise control of microtubule stability and dynamics, achieved through tubulin post-translational modifications and microtubule-associated proteins, is essential for various cellular functions. Dysfunctions in microtubules are strongly implicated in pathogenesis, including neurodegenerative disorders. Ongoing research focuses on microtubule-targeting therapeutic agents that modulate stability, offering potential treatment options for these diseases and cancers. Consequently, understanding the dynamic state of microtubules is crucial for assessing disease progression and therapeutic effects. Traditionally, microtubule dynamics have been assessed in vitro or in cultured cells through rough fractionation or immunoassay, using antibodies targeting post-translational modifications of tubulin. However, accurately analyzing tubulin status in tissues using such procedures poses challenges. In this study, we developed a simple and innovative microtubule fractionation method to separate stable microtubules, labile microtubules, and free tubulin in mouse tissues. The procedure involved homogenizing dissected mouse tissues in a microtubule-stabilizing buffer at a 19:1 volume ratio. The homogenates were then fractionated through a two-step ultracentrifugation process following initial slow centrifugation (2,400 × g) to remove debris. The first ultracentrifugation step (100,000 × g) precipitated stable microtubules, while the resulting supernatant was subjected to a second ultracentrifugation step (500,000 × g) to fractionate labile microtubules and soluble tubulin dimers. This method determined the proportions of tubulin constituting stable or labile microtubules in the mouse brain. Additionally, distinct tissue variations in microtubule stability were observed that correlated with the proliferative capacity of constituent cells. These findings highlight the significant potential of this novel method for analyzing microtubule stability in physiological and pathological conditions.

The bacterial tubulin homolog FtsZ generates electrical oscillations

FtsZ, a major cytoskeletal protein in all bacteria and archaea, forms a ring that directs cytokinesis. Bacterial FtsZ is considered the ancestral homolog of the eukaryotic microtubule (MT)-forming tubulins, sharing GTPase activity and the ability to assemble into protofilaments, rings, and sheets, but not MTs. Previous studies from our laboratory demonstrated that structures of isolated brain MTs spontaneously generate electrical oscillations and bursts of electrical activity similar to action potentials. No information about whether the prokaryotic tubulins may share similar properties is available. Here, we obtained by ammonium sulfate precipitation an enriched protein fraction of the endogenous FtsZ from wild-type Escherichia coli ATCC 25922 without any transfection or overexpression of the protein. As revealed by electron microscopy, FtsZ was detected by dot blot analysis and immunofluorescence that assembled into filaments and sheets in a polymerization buffer. We used the patch-clamp technique to explore the electrical properties of sheets of FtsZ and bacterial cells. Electrical recordings at various holding potentials ranging from ±200 mV showed a complex oscillatory behavior, with several peak frequencies between 12 and 110 Hz in the power spectra and a linear mean current response. To confirm the oscillatory electrical behavior of FtsZ we also conducted experiments with commercial recombinant FtsZ, with similar results. We also detected, by local field potentials, similar electrical oscillations in K+-depolarized pellets of E. coli cultures. FtsZ oscillations had a wider range of frequency peaks than MT sheets from eukaryotic origin. The findings indicate that the bacterial cytoskeleton generates electrical oscillators that may play a relevant role in cell division and unknown signaling mechanisms in bacterial populations.

The electrical properties of isolated microtubules

This study examines the electrical properties of isolated brain microtubules (MTs), which are long hollow cylinders assembled from αβ-tubulin dimers that form cytoskeletal structures engaged in several functions. MTs are implicated in sensory functions in cilia and flagella and cellular activities that range from cell motility, vesicular traffic, and neuronal processes to cell division in the centrosomes and centrioles. We determined the electrical properties of the MTs with the loose patch clamp technique in either the presence or absence of the MT stabilizer Paclitaxel. We observed electrical oscillations at different holding potentials that responded accordingly in amplitude and polarity. At zero mV in symmetrical ionic conditions, a single MT radiated an electrical power of 10–17 W. The spectral analysis of the time records disclosed a single fundamental peak at 39 Hz in the Paclitaxel-stabilized MTs. However, a richer oscillatory response and two mean conductances were observed in the non-Paclitaxel MTs. The findings evidence that the brain MTs are electrical oscillators that behave as "ionic-based" transistors to generate, propagate, and amplify electrical signals.

CROCIN AS NEUROPROTECTIVE AGONIST IN OXIDATIVE DAMAGE AND COGNITIVE DYSFUNCTION INDUCED BY ALUMINIUM CHLORIDE AND COLCHICINE IN RODENTS

The main constituent of saffron, Crocus sativus L., is called Crocin (CR). Water-soluble carotenoids in CR possesses anti-inflammatory, analgesic, anti-edematous, as well as anti-oxidant properties, as well as to increase the number of microtubules in sheep brain microtubules and to have neuroprotective effects. For example, Alzheimer's disease (AD), where the harm of hippocampal as well as cortical neurons results in memory as well as cognitive impairment, as well as amyotrophic lateral sclerosis (ALS), where weakness in muscle is brought on by the degeneration of spinal, bulbar, as well as cortical motor neurons, are disorders which are characterized by the progressive and irreversible loss of neurons. In the current investigation, we evaluated CR's ability to protect neurons from the oxidative harm that Colchicine (Col) and Aluminum chloride (ALCL) induce. GSH level, SOD, besides catalase reduced in the Col model's Col-treated group but was practically returned to normal in the group that received CR. When given CR instead of Col, the levels of MDA, acetylcholine esterase, and nitric oxide returned to nearly normal levels. However, in the Morris water maze test, CR significantly reduces the time it takes to reach the platform (escape latency time), indicating learning and cognitive improvement. Corresponding to this, in the passive avoidance test, the transfer delay time did not significantly increase in the CR-treated group but it did in the Col-treated group. Similar outcomes in the aluminium chloride model were attained. Thus, the present study comes to the conclusion that CR can be utilized to treat oxidative stress and illnesses linked to cognitive dysfunction, such as AD as well as Parkinson’s diseases shown against rodents resultantly. KEYWORDS: Crocin, Aluminium Chloride, Alzheimer’s disease, oxidative stress, Neurodegeneration

Brain Microtubule Electrical Oscillations-Empirical Mode Decomposition Analysis.

Microtubules (MTs) are essential cytoskeletal polymers of eukaryote cells implicated in various cell functions, including cell division, cargo transfer, and cell signaling. MTs also are highly charged polymers that generate electrical oscillations that may underlie their ability to act as nonlinear transmission lines. However, the oscillatory composition and time-frequency differences of the MT electrical oscillations have not been identified. Here, we applied the Empirical Mode Decomposition (EMD) to bovine brain MT sheet recordings to determine the number and fundamental frequencies of the Intrinsic Modes Functions (IMF) and evaluate their energetic contribution to the electrical signal. As previously reported, raw signals were obtained from cow brain MTs (Cantero et al. Sci Rep 6:27143, 2016), sampled, filtered, and subjected to signal decomposition from representative experiments. Filtered signals (200Hz) allowed us to identify either six or seven IMFs. The reconstructed tracings faithfully resembled the original signals, with identifiable frequency peaks. To extend the analysis to obtain time-frequency information and the energy implicated in each IMF, we applied the Hilbert-Huang Transform (HHT) and the Continuous Wavelet Transform (CWT) to the same samples. The analyses disclosed the presence of more fundamental frequency peaks than initially reported and evidenced the advantages and disadvantages of each transform. The study indicates that the EMD is a robust approach to quantifying signal decomposition of brain MT oscillations and suggests novel similarities with human brain wave electroencephalogram (EEG) recordings. The evidence points to the potentially fundamental role of MT oscillations in brain electrical activity.

Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end

In the present study, we used interference reflection microscopy (IRM) to observe the in vitro growth rates and fluctuations of bovine brain microtubules at high temporal resolution. We performed measurements in the presence of GTP and in the presence of the hydrolysis-resistant GTP analog GMPCPP. We fit a computational to the resulting measurements to provide insight into the molecular interactions governing microtubule elongation. Microtubules growing with GMPCPP showed a very low apparent critical concentration, reflecting high affinity interactions between αβ-tubulin subunits and the microtubule end.

Comparative preclinical evaluation of a microtubule PET tracer with a beta‐amyloid PET tracer in AD mice

AbstractBackgroundCurrently available PET tracers used in Alzheimer's disease (AD) have limited utility in prognosis and in revealing reliable clinicopathologic correlations of disease. Therefore, identification and validation of novel biomarkers for AD progression and preclinical disease onset through noninvasive neuroimaging is a top priority in biomedical research. We proposed to target microtubules (MTs) as an imaging target for AD in preclinical, postmortem and clinical studies that support the implication of altered regulation of MTs in AD and positively correlated with neurodegeneration process. Furthermore, MT loss in AD is contributed to a large number of mutated proteins, enzymes, and post‐translational modifications compared to other imaging targets. Therefore, targeting brain MTs may be advantageous, as cumulative loss of MTs is the final common pathway for a variety of biochemical pathologies leading to neurodegeneration in AD. [11C]MPC‐6827 is the pioneer BBB‐penetrating PET tracer available for in vivo imaging of MTs in brain.1,2 Herein, we report the comparative binding of [11C]MPC‐6827, and [11C]PiB in Aβ over expressing APP mutated transgenic J20 mice and controls.Method[11C]MPC‐6827 and [11C]PiB were synthesized using GE Tracerlab FX2 MEI and FX2N modules. Dynamic PET scans were performed for 30 min after tail vein injection of [11C]MPC‐6827 and [11C]PiB in 6‐month‐old J20 mice and littermates (n=4) using a Siemens Focus 220 microPET scanner. Image analyses were performed with Mango software on reconstructed data.ResultPET image analyses show ∼30% of reduced whole brain uptake of [11C]MPC‐6827 in J20 mice whereas, [11C]PiB exhibited modest higher binding (∼10%) in J20 than control mice. Standardized uptake values (SUVs) show similar trend of binding of tracers in prefrontal cortex (PFC) and hippocampus in J20 and control mice.ConclusionOur preliminary studies show that in J20 mice, binding of the [11C]MPC‐6827 is reduced in whole brain, hippocampus and PFC compared control mice and is inversely correlated with [11C]PiB binding. The effect size of [11C]MPC‐6827 seems higher than [11C]PiB. Therefore, [11C]MPC‐6827 could be used as a PET tracer for human brain imaging of AD and other NDs. Acknowledgement: This work was funded by Center for Biomedical Neuroscience (CBN) 2020‐2021, UT Health San Antonio, Texas, USA.

Honeybee Brain Oscillations Are Generated by Microtubules. The Concept of a Brain Central Oscillator.

Microtubules (MTs) are important structures of the cytoskeleton in neurons. Mammalian brain MTs act as biomolecular transistors that generate highly synchronous electrical oscillations. However, their role in brain function is largely unknown. To gain insight into the MT electrical oscillatory activity of the brain, we turned to the honeybee (Apis mellifera) as a useful model to isolate brains and MTs. The patch clamp technique was applied to MT sheets of purified honeybee brain MTs. High resistance seal patches showed electrical oscillations that linearly depended on the holding potential between ± 200 mV and had an average conductance in the order of ~9 nS. To place these oscillations in the context of the brain, we also explored local field potential (LFP) recordings from the Triton X-permeabilized whole honeybee brain unmasking spontaneous oscillations after but not before tissue permeabilization. Frequency domain spectral analysis of time records indicated at least two major peaks at approximately ~38 Hz and ~93 Hz in both preparations. The present data provide evidence that MT electrical oscillations are a novel signaling mechanism implicated in brain wave activity observed in the insect brain.

In vivo evaluation of a microtubule PET ligand, [11C]MPC-6827, in mice following chronic alcohol consumption.

Excessive alcohol consumption is a global health burden and requires a better understanding of its neurobiology. A lower density of brain microtubules is found in alcohol-related human brain disease postmortem and in rodent models of chronic alcohol consumption. Here, we report in vivo imaging studies of microtubules in brain using our recently reported Positron Emission Tomography (PET) tracer, [11C]MPC-6827, in chronic alcohol-consuming adult male C57BL/6J mice and control mice. In vivo PET imaging studies of [11C]MPC-6827 (3.7 ± 0.8MBq) were performed in two groups of adult male mice: (1) water-consuming control mice (n = 4) and (2) mice that consumed 20% alcohol (w/v) for 4months using the intermittent 2-bottle choice procedure that has been shown to lead to signs of alcohol dependence. Dynamic 63min PET images were acquired using a microPET Inveon system (Siemens, Germany). PET images were reconstructed using the 3D-OSEM algorithm and analyzed using VivoQuant version 4 (Invicro, MA). Tracer uptake in ROIs that included whole brain, prefrontal cortex (PFC), liver and heart was measured and plotted as %ID/g over time (0-63min) to generate time-activity curves (TACs). In general, a trend for lower binding of [11C]MPC-6827 in the whole brain and PFC of mice in the chronic alcohol group was found compared with control group. No group difference in radiotracer binding was found in the peripheral organs such as liver and heart. This pilot study indicates a trend of loss of microtubule binding in whole brain and prefrontal cortex of chronic alcohol administered mice brain compared to control mice, but no loss in heart or liver. These results indicate the potential of [11C]MPC-6827 as a PET ligand for further in vivo imaging investigations of AUD in human.

Electrical behaviour and evolutionary computation in thin films of bovine brain microtubules

We report on the electrical behaviour of thin films of bovine brain microtubules (MTs). For samples in both their dried and hydrated states, the measured currents reveal a power law dependence on the applied DC voltage. We attribute this to the injection of space-charge from the metallic electrode(s). The MTs are thought to form a complex electrical network, which can be manipulated with an applied voltage. This feature has been exploited to undertake some experiments on the use of the MT mesh as a medium for computation. We show that it is possible to evolve MT films into binary classifiers following an evolution in materio approach. The accuracy of the system is, on average, similar to that of early carbon nanotube classifiers developed using the same methodology.

Green Lithography for Delicate Materials

AbstractA variety of unconventional materials, including biological nanostructures, organic and hybrid semiconductors, as well as monolayer, and other low‐dimensional systems, are actively explored. They are usually incompatible with standard lithographic techniques that use harsh organic solvents and other detrimental processing. Here, a new class of green and gentle lithographic resists, compatible with delicate materials and capable of both top‐down and bottom‐up fabrication routines is developed. To demonstrate the excellence of this approach, devices with sub‐micron features are fabricated on organic semiconductor crystals and individual animal's brain microtubules. Such structures are created for the first time, thanks to the genuinely water‐based lithography, which opens an avenue for the thorough research of unconventional delicate materials at the nanoscale.

Electrical Oscillations of Isolated Brain Microtubules

Microtubules (MTs) are important cytoskeletal structures engaged in a number of specific cellular activities, including vesicular traffic and cell division, as well as information transfer within neuronal processes. MTs also are highly charged polyelectrolytes. Recent in vitro electrophysiological studies indicate that different brain MT assemblies, including two-dimensional (2D) sheets (MT sheets) and macrotube bundles, generate highly synchronous electrical oscillations (Cantero et al. Sci Rep, 2016 & 2018). Although taxol-stabilized isolated MTs are capable of amplifying electrical signals, no information is heretofore available as to whether isolated they also engage in electrical oscillations. Herein we tested the effect of voltage clamping on the electrical properties of non-taxol stabilized isolated brain MTs. Electrical oscillations were observed at holding potentials between ±200 mV. Mean oscillatory currents were linear with respect to holding potential, with a change in conductance from 59.6 ± 3.6 nS to 160.8 ± 7.6 nS (n = 3) after loose-patch correction. The average change in conductance was much higher than previously reported for more complex MT structures. Spectral analysis of the electrical currents also disclosed a richer oscillatory response as compared to voltage clamped MT sheets from the same preparation. The findings are consistent with the possibility that MT assemblies (i.e. bundles, sheets) may render more coherent responses at given oscillatory frequencies such as entraining to oscillate together. The oscillatory behavior of isolated brain MTs is consistent with that of “ionic-based” transistors whose activity is synchronized in higher MT structures. The ability of MTs to generate, propagate, and amplify electrical signals may have important implications in neuronal computational capabilities.

The long-term effects of Δ9-tetrahydrocannabinol on microtubule dynamicity in rats

Studies reported that Δ9-tetrahydrocannabinol (Δ9-THC) is an essential drug as an anti-cancer, neuroprotective, anti-inflammatory, and immune-modulatory agent. However, the mechanism by which Δ9-THC causes these events remains to be elucidated. We attempted to investigate the in vivo studies of Δ9-THC on brain microtubule dynamicity, and acetylcholinesterase (AChE) activity. The microtubule polymerization, secondary and tertiary structures of α/β-tubulins, as well as the AChE activity, were evaluated in the experimental groups. The significantly lowest optical density and initial rate of polymerization was observed in THC 3 mg/kg, THC 9 mg/kg, and THC 18 mg/kg treated groups. The content of secondary and tertiary structures of α/β-tubulins was significantly affected in treated groups. The AChE activity was significantly lower in treated groups in a dose-dependent manner. These data highlight the microtubule dynamicity as a molecular target for Δ9-THC, which affects memory dysfunction. However, Δ9-THC can be inhibited the AChE activity and provide an improved therapeutics for neurodegenerative diseases.

Synthesis and Initial In Vivo Evaluations of [11C]WX‐132‐18B, a Microtubule PET Imaging Agent

AbstractMicrotubules play an important role in brain functioning and purported to alter in several neurological disorders including Alzheimer's disease, Parkinson disease, malignancies and brain injuries. Creating a molecular imaging probe that binds to microtubules in brain would significantly benefit the diagnosis and stratification of patients based on therapeutic treatments that target neurotubulin pathology. We have previously reported the first‐ and second‐generation microtubule Positron Emission Tomography (PET) ligands, which exhibited blood brain barrier permeability and accumulation in brain. We are continuing the search for new microtubule agents in order to establish the most selective PET agent with optimal attributes for in vivo quantification of the brain microtubules in diseases. Hence, we identified a high‐affinity microtubule agent namely, WX‐MB‐18B (GI50= 1.9 nM, IC50=0.45‐0.99 nM). In addition to the high affinity to microtubules, WX132‐18B has adequate lipophilicity for facile brain entry and also is amenable for 11 C‐radiolabeling. Herein we report the radiochemical synthesis and initial PET evaluation of [11C]WX‐132‐18B in mice to determine its ability for imaging brain microtubules.

The Bacterial Tubulin Homolog FtsZ Forms 2D-sheets that Sustain Electrical Oscillations

FtsZ, a major cytoskeletal protein present in all bacteria and archaea forms a cytokinetic ring, the Z ring, at the site of septation, which directs cytokinesis. Multiple sequence alignment and secondary structure predictions indicate that bacterial FtsZ is a homolog of the eukaryotic tubulins which, as their mammalian counterpart, binds guanine nucleotides, has GTPase activity, and is able to assemble into protofilamental structures in a GTP-dependent manner. Previous studies from our laboratory demonstrated that microtubule (MTs) structures isolated from murine, bovine and apian brains spontaneously generate electrical oscillations and bursts of electrical activity similar to action potentials. Although the electrical behavior of brain MTs may have important implication in eukaryotic cell function, no information is available as to whether FtsZ may share similar properties. Therefore, the aim of the present study was to explore whether FtsZ protofilamental sheets may sustain electrical activity. FtsZ protein from E. coli ATCC 25922 was purified by ammonium sulfate precipitation, and assembled into protofilamental sheets by incubation with a polymerization buffer. 2D FtsZ sheets were voltage-clamped, and gigaseals obtained in most experiments (n = 7). Subsequently, electrical recordings from output currents were obtained, showing a complex oscillatory behavior with several peak frequencies between 7 and 110 Hz in the power spectra. As a result, it was observed that FtsZ 2D sheets had a much wider range of frequencies compared to MTs sheets from eukaryotic origin. The findings demonstrate that FtsZ protofilamental sheets are capable of generating electrical signals and behave as electrical oscillators that may be relevant to the cell division process and/or yet unknown signaling mechanisms.

Brain Microtubule Structures Behave as Memristive Devices

Brain Microtubule Structures Behave as Memristive Devices

Actin filaments modulate electrical activity of brain microtubule protein two-dimensional sheets.

The cytoskeleton of eukaryotic cells contains networks of actin filaments and microtubules (MTs) that are jointly implicated in various cell functions, including cell division, morphogenesis, and migration. In neurons, this synergistic activity drives both the formation of axons during development and synaptic activity in mature neurons. Both actin filaments and MTs also are highly charged polyelectrolytes that generate and conduct electrical signals. However, no information is presently available on a potential electrical crosstalk between these two cytoskeletal networks. Herein we tested the effect of actin polymerization on the electrical oscillations generated by two-dimensional sheets of bovine brain microtubule protein (2D-MT). The voltage-clamped 2D-MT sheets displayed spontaneous electrical oscillations representing a synchronous 224% change in conductance, and a fundamental frequency of 38 Hz. At 60 mV, a 4.15 nC of integrated charge transferred per second increased by 72.3% (7.15 nC) after addition of monomeric (G)-actin. This phenomenon had a 2-min lag time, and was prevented by the presence of the G-actin-binding protein DNAse I. Addition of prepolymerized F-actin, however, had a rapid onset (<10 s) and a higher effect on the tubulin sheets (~100% increase, 8.25 nC). The data are consistent with an interaction between the actin cytoskeleton and tubulin structures, in what seems to be an electrostatic effect. Because actin filaments and MTs interact with each other in neurons, it is possible for this phenomenon to be present, and of relevance in the processing of intracellular signaling, including the gating and activation of actin cytoskeleton-regulated excitable ion channels in neurons.

In vivo comparison of N-11CH3 vs O-11CH3 radiolabeled microtubule targeted PET ligands

Altered dynamics of microtubules (MT) are implicated in the pathophysiology of a number of brain diseases. Therefore, radiolabeled MT targeted ligands that can penetrate the blood brain barrier (BBB) may offer a direct and sensitive approach for diagnosis, and assessing the clinical potential of MT targeted therapeutics using PET imaging. We recently reported two BBB penetrating radioligands, [11C]MPC-6827 and [11C]HD-800 as specific PET ligands for imaging MTs in brain. The major metabolic pathway of the above molecules is anticipated to be via the initial labeling site, O-methyl, compared to the N-methyl group. Herein, we report the radiosynthesis of N-11CH3-MPC-6827 and N-11CH3-HD-800 and a comparison of their in vivo binding with the corresponding O-11CH3 analogues using microPET imaging and biodistribution methods. Both O-11CH3 and N-11CH3 labeled MT tracers exhibit high specific binding and brain. The N-11CH3 labeled PET ligands demonstrated similar in vivo binding characteristics compared with the corresponding O-11CH3 labeled tracers, [11C]MPC-6827 and [11C]HD-800 respectively.

Comparison of polymerization and structural behavior of microtubules in rat brain and sperm affected by the extremely low-frequency electromagnetic field

BackgroundMicrotubule proteins are able to produce electromagnetic fields and have an important role in memory formation, and learning. Therefore, microtubules have the potential to be affected by exogenous electromagnetic fields. This study aimed to examine the comparison of microtubule polymerization and its structural behavior in brain and sperm affected by 50 Hz extremely low-frequency electromagnetic field (ELEF).ResultsTwenties adult male rats were randomly and equally divided into control and experimental groups, to evaluate the effect of 50 Hz ELEF on the sperm and brain functions. Plus-maze, serum testosterone and corticosterone, and sperm evaluation were performed. Next, the semen and brain samples were obtained, and they were divided into four experimental groups for investigation of microtubule polymerization. There was no significant difference in testosterone and, corticosterone levels, anxiety behaviors, and sperm morphology between control and ELEF-exposure groups. The sperm viability, total and progressive motility were significantly higher in the ELEF-exposed group than that of the control group. The microtubule polymerization in sperm ELEF was significantly higher than in other groups. The secondary and tertiary structures of tubulins were significantly affected in the brain, and sperm ELEF groups.ConclusionIt seems that the polymerization of microtubules and conformational changes of tubulin dimers are improved by ELEF application.