Neuronal Tubulin Research Articles

P-tau Ser356 is associated with Alzheimer’s disease pathology and is lowered in brain slice cultures using the NUAK inhibitor WZ4003

Tau hyperphosphorylation and aggregation is a common feature of many dementia-causing neurodegenerative diseases. Tau can be phosphorylated at up to 85 different sites, and there is increasing interest in whether tau phosphorylation at specific epitopes, by specific kinases, plays an important role in disease progression. The AMP-activated protein kinase (AMPK)-related enzyme NUAK1 has been identified as a potential mediator of tau pathology, whereby NUAK1-mediated phosphorylation of tau at Ser356 prevents the degradation of tau by the proteasome, further exacerbating tau hyperphosphorylation and accumulation. This study provides a detailed characterisation of the association of p-tau Ser356 with progression of Alzheimer’s disease pathology, identifying a Braak stage-dependent increase in p-tau Ser356 protein levels and an almost ubiquitous presence in neurofibrillary tangles. We also demonstrate, using sub-diffraction-limit resolution array tomography imaging, that p-tau Ser356 co-localises with synapses in AD postmortem brain tissue, increasing evidence that this form of tau may play important roles in AD progression. To assess the potential impacts of pharmacological NUAK inhibition in an ex vivo system that retains multiple cell types and brain-relevant neuronal architecture, we treated postnatal mouse organotypic brain slice cultures from wildtype or APP/PS1 littermates with the commercially available NUAK1/2 inhibitor WZ4003. Whilst there were no genotype-specific effects, we found that WZ4003 results in a culture-phase-dependent loss of total tau and p-tau Ser356, which corresponds with a reduction in neuronal and synaptic proteins. By contrast, application of WZ4003 to live human brain slice cultures results in a specific lowering of p-tau Ser356, alongside increased neuronal tubulin protein. This work identifies differential responses of postnatal mouse organotypic brain slice cultures and adult human brain slice cultures to NUAK1 inhibition that will be important to consider in future work developing tau-targeting therapeutics for human disease.

Loss of tubulin tyrosine ligase, a novel feature of sporadic and familial AD, is an enhancer of tau phosphorylation

AbstractBackgroundWe have recently shown that loss of tubulin tyrosine ligase (TTL) and subsequent disruption of a‐tubulin retyrosination is a feature of AD and a promoter of synaptic loss by inhibiting microtubule entries into dendritic spines. TTL+/‐ mice have cognitive deficits, altered LTP, synapse loss and increased levels of detyrosinated and D2 tubulin, two tubulin modifications that accumulate on non‐dynamic microtubules. We also found that low levels of phospho‐tau correlated with higher levels of detyrosinated and D2 tubulin in pyramidal neurons residing in the anterior hippocampal formation of human AD brains, raising the question of whether improper microtubule longevity drives early stages of tau hyperphosphorylation in AD. Here, we demonstrate that loss of TTL enhances both AMPK activation and tau phosphorylation in cultured rat hippocampal and human cortical neurons, and present preliminary evidence on the mechanisms by which this regulation may occur and contribute to TTL‐loss dependent synaptic dysfunction.MethodHuman cortical neurons derived from iPSC lines in which the London familial APP mutation V717I was knocked into one allele of the IMR90 control using CRISPR/Cas9, were used as a cellular model of familial AD. Lentiviral infection with shRNA against TTL was used to silence TTL expression. Microtubule dynamics were measured by confocal time‐lapse microscopy in neurons expressing EB3‐EGFP, a protein that tracks the growing plus ends of microtubules. Immunoblotting was performed to measure both unmodified and modified levels of TLL, tubulins, AMPK subunits, total tau, p262 and AT8 phospho‐tau.ResultIn the APP neurons, we found that a reduction in TTL levels correlated with an increase in p262/AT8 phospho‐tau variants and phospho‐AMPK, the active state of one of the kinases that phosphorylates serine 262, a key residue in the binding of tau to microtubules. Knockdown of TTL was sufficient to reduce microtubule dynamics and increase both active AMPK levels and phospho‐tau.ConclusionOur results indicate that loss of TTL alone reduces dynamic microtubules, triggers tau kinase activation, and results in hyperphosphorylated tau. Together with our previously published data, these findings suggest that reduced levels of TTL act as one of the early drivers of tau hyperphosphorylation in AD.

HIV-1 gp120-Induced Cognitive Disorder Is Concomitant with Altered Gut Microbiota

Background HIV-associated neurocognitive disorders (HAND) is an essential factor affecting the quality of life of people living with HIV (PLWH), and the prevalence of HAND in PLWH can reach 19% to 64%. More than 50% of HAND patients treated with highly active antiretroviral therapy (HAART) did not show improvement in symptoms of cognitive impairment. HIV-1 envelope protein gp120 may be a major contributor to HAND pathology. HIV-1 gp120 enters neurons by binding CXCR4 and CCR5 and can also bind neuronal tubulin to cause direct neurotoxicity and neuronal death. HIV-1 gp120 transgenic mice (Tgm) are commonly used as HAND animal models, and Tgm brain pathological changes and differential gene expression are highly consistent with HAND patients. Intestinal flora affects the progression of diseases by regulatory the intestinal barrier, and several studies have confirmed that the normalization of abnormal intestinal flora reduces the damage to the nervous system in a variety of neurodegenerative diseases. Studies reported that Akkermansia muciniphila has a downward trend in PLWH, which may cause intestinal barrier damage. There is still a lack of relevant evidence to confirm the association between abnormal changes in gut microbiota and HAND induced by HIV-1 gp120. Therefore, in this study, we examined the gut microbiota changes in 6, 9, and 12 months Tgm to explore the relationship between age-dependent significantly different bacterial genera, intestinal barrier and neuronal damage. Methods The fresh feces were collected from HIV-1 gp120 transgenic mice (Tgm) and C57BL/6J mice (wild type, WT) at ages 6, 9, and 12 months using perianal stimulation at the same time (n=5). Total bacterial DNA was extracted from each fecal sample and the V3-V4 region of 16S rRNA was sequenced by the Illumina MiSeq platform. 12 months Tg and WT mice were sacrificed, and serum was collected to detect lipopolysaccharide (LPS) concentration by ELISA, and brain tissue of mice in each group was collected for Map2 immunohistochemical staining (n=5). SPSS 20.0 software was used for statistical analysis. P <0.05 were considered statistically significant. Results 16S rRNA analysis showed that increased age was significantly associated with increased abundance of Blautia ( P <0.01) and Prevotellaceae_NK3B31_group ( P <0.0001) genus and decreased abundance of Akkermansia genus ( P <0.05) in Tgm. Notably, the increasing age of WT was associated with the opposite trend of Blautia, Prevotellaceae_NK3B31_group and Akkermansia genus abundance, indicating those genus changes with age and gp120 induced. To further explore the effect of gut microbiota on the gut barrier, serum LPS concentrations were detected for 12 months Tgm and WT. The results showed that serum LPS level was significantly higher in Tgm than in WT ( P <0.01), indicating that the intestinal barrier may be impaired in Tgm. Finally, we collected 12 months Tgm and WT brain tissues for Map2 immunohistochemical staining to evaluate neuronal synaptic length and integrity. Compared with WT, the expression of map2 in the hippocampus and cerebral cortex was significantly decreased ( P <0.05), indicating neuronal damage in Tgm. Conclusion At the genus level, the abundance of Akkermansia was negatively correlated and the Blautia and Prevotellaceae_NK3B31_group abundance was positively correlated with increasing age in Tgm but showed an opposite trend in WT. The serum LPS level increased dramatically in the 12 months Tgm, indicating Tgm with intestinal barrier damage. Map2 expression was reduced considerably hippocampus and cortex in Tgm brain, which means neuronal synaptic integrity was impaired. ( Acknowledgements: National Natural Science Foundation of China, No. 82172259 to H.C.; Corresponding author: Hong Cao, gzhcao@smu.edu.cn.)

Comparison of the Molecular Motility of Tubulin Dimeric Isoforms: Molecular Dynamics Simulations and Diffracted X-ray Tracking Study.

Tubulin has been recently reported to form a large family consisting of various gene isoforms; however, the differences in the molecular features of tubulin dimers composed of a combination of these isoforms remain unknown. Therefore, we attempted to elucidate the physical differences in the molecular motility of these tubulin dimers using the method of measurable pico-meter-scale molecular motility, diffracted X-ray tracking (DXT) analysis, regarding characteristic tubulin dimers, including neuronal TUBB3 and ubiquitous TUBB5. We first conducted a DXT analysis of neuronal (TUBB3-TUBA1A) and ubiquitous (TUBB5-TUBA1B) tubulin dimers and found that the molecular motility around the vertical axis of the neuronal tubulin dimer was lower than that of the ubiquitous tubulin dimer. The results of molecular dynamics (MD) simulation suggest that the difference in motility between the neuronal and ubiquitous tubulin dimers was probably caused by a change in the major contact of Gln245 in the T7 loop of TUBB from Glu11 in TUBA to Val353 in TUBB. The present study is the first report of a novel phenomenon in which the pico-meter-scale molecular motility between neuronal and ubiquitous tubulin dimers is different.

Abstract P078: Cancer cell derived extracellular vesicles convey protein signatures of small cell lung carcinoma

Abstract Extracellular vesicles (EVs) are known effectors of cell signaling and exchange implicated in cancer development and progression, as well as remodeling and immunomodulation of the tumor microenvironment. EVs are detected in various biofluids and are considered to convey signature features of the cells from which they originate. Clinical assessment of plasma-derived EV molecular contents therefore offers promise of “liquid biopsies” for detection or subtyping of cancers as well as dynamic reporting of tumor status during and after treatment. To date, there have been no comprehensive analyses aimed at interrogating the diverse repertoire of EV protein cargoes in small cell lung carcinoma (SCLC). Using plasmas from a cohort of 18 SCLC cases, 16 lung adenocarcinoma (LUAD) cases and 34 controls matched based on smoking status, age, and sex, EVs were enriched via affinity capture using magnetic beads functionalized with phosphatidyl serine binding protein TIM4 in addition to single-step density flotation ultracentrifugation to deplete abundant protein background. In-depth proteomic profiling was performed on enriched EVs via nano liquid chromatography / high-resolution ion-mobility mass spectrometry. Profiles were intersected with proteomic profiles generated from intratumor EVs dissociated from 12 SCLC patient-derived xenograft (PDX) mouse models as well as EVs isolated from cell-conditioned medias of a panel of 17 SCLC cell lines. A total of 496 proteins were quantified in TIM4pos EVs. Unsupervised hierarchical clustering and principal component analyses showed that TIM4pos EV protein cargoes differentiate SCLC from LUAD and matched controls. Differential analyses identified 117 proteins that were elevated (AUC> 0.60) in TIM4pos SCLC EVs compared to controls. Of these 117 features, 101 (86%) were also elevated (AUC> 0.60) in TIM4pos SCLC EVs compared to TIM4pos LUAD EVs. Intersection of proteins elevated in TIM4pos SCLC EVs with proteomic profiles of intratumor EVs from SCLC PDX models and conditioned media-derived EVs from SCLC cell lines yielded 67 and 92 overlapping proteins, respectively. Overlapping proteins were characterized by YAP1-associated signatures of cytoskeletal re-arrangement and epithelial-to-mesenchymal transition and included a prevalence of neuronal tubulin and actin family members, YWHA family members, and annexins. We developed and applied an optimized EV isolation and proteomics workflow to profile EV protein cargoes and establish circulating EV-associated protein signatures of SCLC with biomarker potential. Our integrated analyses identified novel EV-associated proteins for detection of SCLC that are associated with oncogenic drivers. These studies demonstrate that EVs harbor cancer cell disseminated signatures and that continued efforts to query the EV proteome towards discovery of novel SCLC biomarkers are warranted. Citation Format: Jody Vykoukal, Taketo Kato, Hiroyuki Katayama, Allison Stewart, Ehsan Irajizad, Yining Cai, Fu-Chung Hsiao, Jennifer B. Dennison, Edwin J. Ostrin, Hai T. Tran, Carl M. Gay, Lauren A. Byers, Johannes F. Fahrmann, Samir M. Hanash. Cancer cell derived extracellular vesicles convey protein signatures of small cell lung carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P078.

Nano-positioning and tubulin conformation contribute to axonal transport regulation of mitochondria along microtubules

Correct spatiotemporal distribution of organelles and vesicles is crucial for healthy cell functioning and is regulated by intracellular transport mechanisms. Controlled transport of bulky mitochondria is especially important in polarized cells such as neurons that rely on these organelles to locally produce energy and buffer calcium. Mitochondrial transport requires and depends on microtubules that fill much of the available axonal space. How mitochondrial transport is affected by their position within the microtubule bundles is not known. Here, we found that anterograde transport, driven by kinesin motors, is susceptible to the molecular conformation of tubulin in neurons both invitro and invivo. Anterograde velocities negatively correlate with the density of elongated tubulin dimers like guanosine triphosphate (GTP)-tubulin. The impact of the tubulin conformation depends primarily on where a mitochondrion is positioned, either within or at the rim of microtubule bundle. Increasing elongated tubulin levels lowers the number of motile anterograde mitochondria within the microtubule bundle and increases anterograde transport speed at the microtubule bundle rim. We demonstrate that the increased kinesin velocity and density on microtubules consisting of elongated dimers add to the increased mitochondrial dynamics. Our work indicates that the molecular conformation of tubulin contributes to the regulation of mitochondrial motility and as such to the local distribution of mitochondria along axons.

Crosstalk between acetylation and the tyrosination/detyrosination cycle of α-tubulin in Alzheimer’s disease

Microtubules (MTs) support a variety of neuronal functions, such as maintenance of cell structure, transport, and synaptic plasticity. Neuronal MTs are highly heterogeneous due to several tubulin isotypes and the presence of multiple post-translational modifications, such as detyrosination and acetylation. The tubulin tyrosination/detyrosination cycle is a key player in the maintenance of MT dynamics, as tyrosinated tubulin is associated with more dynamic MTs, while detyrosinated tubulin is linked to longer lived, more stable MTs. Dysfunction of tubulin re-tyrosination was recently correlated to Alzheimer’s disease progression. The implication of tubulin acetylation in Alzheimer’s disease has, however, remained controversial. Here, we demonstrate that tubulin acetylation accumulates in post-mortem brain tissues from Alzheimer’s disease patients and human neurons harboring the Alzheimer’s familial APP-V717I mutation. We further show that tubulin re-tyrosination, which is defective in Alzheimer’s disease, can control acetylated tubulin in primary neurons irrespective of the levels of the enzymes regulating tubulin acetylation, suggesting that reduced MT dynamics associated with impaired tubulin re-tyrosination might contribute to the accumulation of tubulin acetylation that we detected in Alzheimer’s disease.

Generation of an induced pluripotent stem cell line (DHMCi008-A) from an individual with TUBA1A tubulinopathy

Variants in different neuronal tubulin isotypes cause severe neurodevelopmental disorders with cerebral malformations accompanied by developmental delay, motor impairment, and epilepsy, known as tubulinopathies. Induced pluripotent stem cells were generated from peripheral blood mononuclear cells from a female subject carrying the heterozygous de novo variant c.[521C > T] (p.[Ala174Val]) in the TUBA1A gene. PBMCs were reprogrammed using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Invitrogen) and showed a normal karyotype, expression of pluripotency markers, and spontaneous in vitro differentiation into all three germ layers. The generated iPSCs represent a useful tool to study the pathophysiology of TUBA1A tubulinopathy.

Coevolution study of tau and a-synuclein suggests a connection between their normal interaction in neurons and the Parkinson's disease-associated mutation A53T

Alpha-synuclein lies at the center of Parkinson’s disease etiology, and polymorphisms in the gene for the microtubule-associated protein tau are risk factors for getting the disease. Tau and a-synuclein interact in vitro, and a-synuclein can also compete with tau binding to microtubules. To test whether these interactions might be part of their natural biological functions, a correlated mutation analysis was performed between tau and a-synuclein, looking for evidence of coevolution. For comparison, analyses were also performed between tau and b- and g-synuclein. In addition, analyses were performed between tau and the synuclein proteins and the neuronal tubulin proteins. Potential correlated mutations were detected between tau and a-synuclein, one involving an a-synuclein residue known to interact with tau in vitro, Asn122, and others involving the Parkinson’s disease-associated mutation A53T. No significant correlated mutations were seen between tau and b- and g-synuclein. Tau showed potential correlated mutations with the neuron-specific bIII-tubulin protein, encoded by the TUBB3 gene. No convincing correlated mutations were seen between the synuclein and tubulin proteins, with the possible exception of b-synuclein with bIVa-tubulin, encoded by the TUBB4A gene. While the correlated mutations between tau and a-synuclein suggest the two proteins have coevolved, additional study will be needed to confirm that their interaction is part of their normal biological function in cells.

The Loss of α- and β-Tubulin Proteins Are a Pathological Hallmark of Chronic Alcohol Consumption and Natural Brain Ageing.

Repetitive excessive alcohol intoxication leads to neuronal damage and brain shrinkage. We examined cytoskeletal protein expression in human post-mortem tissue from Brodmann’s area 9 of the prefrontal cortex (PFC). Brain samples from 44 individuals were divided into equal groups of 11 control, 11 alcoholic, 11 non-alcoholic suicides, and 11 suicide alcoholics matched for age, sex, and post-mortem delay. Tissue from alcoholic cohorts displayed significantly reduced expression of α- and β-tubulins, and increased levels of acetylated α-tubulin. Protein levels of histone deacetylase-6 (HDAC6), and the microtubule-associated proteins MAP-2 and MAP-tau were reduced in alcoholic cohorts, although for MAPs this was not significant. Tubulin gene expressions increased in alcoholic cohorts but not significantly. Brains from rats administered alcohol for 4 weeks also displayed significantly reduced tubulin protein levels and increased α-tubulin acetylation. PFC tissue from control subjects had reduced tubulin protein expression that was most notable from the sixth to the eighth decade of life. Collectively, loss of neuronal tubulin proteins are a hallmark of both chronic alcohol consumption and natural brain ageing. The reduction of cytosolic tubulin proteins could contribute to the brain volumetric losses reported for alcoholic patients and the elderly.

Imbalanced Expression of Tau and Tubulin Induces Neuronal Dysfunction in C. elegans Models of Tauopathy.

Tauopathy is a type of dementia defined by the accumulation of filamentous tau inclusions in neural cells. Most types of dementia in the elderly, including Alzheimer’s disease, are tauopathies. Although it is believed that tau protein abnormalities and/or the loss of its functions results in neurodegeneration and dementia, the mechanism of tauopathy remains obscure. Loss of microtubules and/or tubulin is a known consequence of tau accumulating in neurons in Alzheimer’s disease. In other words, there is an excess level of tau relative to tubulin in tauopathy neurons. To test whether this imbalance of tau and tubulin expression results in the neurotoxicity of tau, we developed several transgenic C. elegans lines that express human tau at various levels in pan-neurons. These worms showed behavioral abnormalities in a tau expression-dependent manner. The knockdown of a tubulin-specific chaperon, or a subset of tubulin, led to enhanced tau toxicity even in low-expressing tau-transgenic worms that showed no abnormal behaviors. In addition, the suppression of tau expression in tubulin knockdown worms rescued neuronal dysfunction. Thus, not only the overexpression of tau but also a reduction in tubulin can trigger the neurotoxicity of tau. Tau expressed in worms was also highly phosphorylated and largely bound to tubulin dimers rather than microtubules. Relative amount of tubulin-unbound tau was increased in high-expressing tau-transgenic worms showing tau toxicity. We further demonstrated that tau aggregation was inhibited by co-incubation of purified tubulin in vitro, meaning sufficient amounts of tubulin can protect against the formation of tau inclusions. These results suggest that the expression ratio of tau to tubulin may be a determinant of the tauopathy cascade.

Towards an understanding of the isotype-specific functions of tubulin in neurons: Technical advances in tubulin expression and purification

Microtubules are cytoskeletal filaments critical for determining the complex morphology of neurons, as well as the basic architecture and organization of mitosis in all eukaryotic cells. Microtubules in humans are composed of 8 α- and 9 β-tubulin isotypes, each of which is encoded by different members of a multi-gene family. The expression pattern of tubulin isotypes, in addition to isotype-specific post-translational modifications, is thought to be critical for the morphogenesis of axons and dendrites. Recent studies revealed that several neurodevelopmental disorders are caused by mutations of specific tubulin isotypes, suggesting that each tubulin isotype has distinct functions. Therefore, in vitro and in vivo functional analyses of tubulin isotypes are important to understand the pathogenesis of developmental disorders. Likewise, analysis of developmental disorders may clarify the function of different tubulin isotypes. In this respect, both the preparation of specific tubulin isotypes and of specific mutant tubulin proteins is critical to understanding the function of tubulin. In the last 20 years, various methods have been developed to study functional differences between tubulin isotypes and the functional defects caused by tubulin mutations. These technical achievements have been discussed in this review. The function of tubulin/microtubules in neuronal morphogenesis as revealed through these techniques has also been described.

Spastin regulates VAMP7-containing vesicles trafficking in cortical neurons

Alteration of axonal transport has emerged as a common precipitating factor in several neurodegenerative disorders including Human Spastic Paraplegia (HSP). Mutations of the SPAST (SPG4) gene coding for the spastin protein account for 40% of all autosomal dominant uncomplicated HSP. By cleaving microtubules, spastin regulates several cellular processes depending on microtubule dynamics including intracellular membrane trafficking. Axonal transport is fundamental for the viability of motor neurons which often have very long axons and thus require efficient communication between the cell body and its periphery. Here we found that the anterograde velocity of VAMP7 vesicles, but not that of VAMP2, two vesicular-SNARE proteins implicated in neuronal development, is enhanced in SPG4-KO neurons. We showed that this effect is associated with a slight increase of the level of acetylated tubulin in SPG4-KO neurons and correlates with an enhanced activity of kinesin-1 motors. Interestingly, we demonstrated that an artificial increase of acetylated tubulin by drugs reproduces the effect of Spastin KO on VAMP7 axonal dynamics but also increased its retrograde velocity. Finally, we investigated the effect of microtubule targeting agents which rescue axonal swellings, on VAMP7 and microtubule dynamics. Our results suggest that microtubule stabilizing agents, such as taxol, may prevent the morphological defects observed in SPG4-KO neurons not simply by restoring the altered anterograde transport to basal levels but rather by increasing the retrograde velocity of axonal cargoes.

The viral protein gp120 decreases the acetylation of neuronal tubulin: potential mechanism of neurotoxicity.

The human immunodeficiency virus (HIV) envelope protein gp120 promotes axonal damage and neurite pruning, similar to that observed in HIV-positive subjects with neurocognitive disorders. Thus, gp120 has been used to examine molecular and cellular pathways underlying HIV-mediated neuronal dysfunction. Gp120 binds to tubulin beta III, a component of neuronal microtubules. Microtubule function, which modulates the homeostasis of neurons, is regulated by polymerization and post-translational modifications. Based on these considerations, we tested the hypothesis that gp120 induces dynamic instability of neuronal microtubules. We first observed that gp120 prevents the normal polymerization of tubulin invitro. We then tested whether gp120 alters the post-translational modifications in tubulin by examining the ability of gp120 to change the levels of acetylated tubulin in primary rat neuronal cultures. Gp120 elicited a time-dependent decrease in tubulin acetylation thatwas reversed by Helix-A peptide, a compound that competitively displaces the binding of gp120 to neuronal microtubules. To determine whether post-translational modifications in tubulin also occur invivo, we measured acetylated tubulin in the cerebral cortex of HIV transgenic rats (HIV-tg). We observed a decrease in tubulin acetylation in 5- and 9-month-old HIV-tg rats when compared to age-matched wild type. Neither changes in microglia morphology nor alterations in mRNA levels for interleukin-1β and tumor necrosis factor α were detected in 5-month-old animals. Our findings propose neuronal microtubule instability as a novel mechanism of HIV neurotoxicity, without evidence of enhanced inflammation.

Mechanism of Propofol's Action on MAP Rich Tubulin and Actin - An In Vitro Study

Despite a century of sustained research, brain scientists remain ignorant of the workings of the three pound organ that is the seat of all conscious human activity. Many have tried to attack the problem by examining the nervous systems of simpler organisms. The difficulty in establishing a link between biology and behaviour in humans is still more acute. There are techniques which are used to record the activity of single neurons in living humans. Such breakthrough methods could, in principle, begin to bridge the gap between the firing of neurons and cognition: perception, emotion, decision making and ultimately, consciousness itself. Deciphering the exact patterns of brain activity that underlie thinking and behavior will also provide critical insights into what happens when neural circuitry malfunctions in psychiatric and neurological disorders-schizophrenia, autism, Alzheimer's or Parkinson's. Anesthetics are also known to inhibit neuronal fast anterograde axoplasmic transport (FAAT) in a reversible and dose-dependent manner, but the precise mechanism by which anesthetic prevent consciousness remains unknown largely because the mechanism by which brain physiology produces consciousness is unexplained. In the present study we have used circular dichroism spectroscopy and confocal laser scanning microscopy to see the effect of propofol on the assembly of neuronal tubulin and actin together and probed into the changes of their secondary structures.

Structure and Dynamics of Single-isoform Recombinant Neuronal Human Tubulin

Microtubules are polymers that cycle stochastically between polymerization and depolymerization, i.e. they exhibit “dynamic instability.” This behavior is crucial for cell division, motility, and differentiation. Although studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure, and dynamics has been held back by a lack of dynamics measurements with and structural characterization of homogeneous isotypically pure engineered tubulin. Here, we report for the first time the cryo-EM structure and in vitro dynamics parameters of recombinant isotypically pure human tubulin. α1A/βIII is a purely neuronal tubulin isoform. The 4.2-Å structure of post-translationally unmodified human α1A/βIII microtubules shows overall similarity to that of heterogeneous brain microtubules, but it is distinguished by subtle differences at polymerization interfaces, which are hot spots for sequence divergence between tubulin isoforms. In vitro dynamics assays show that, like mosaic brain microtubules, recombinant homogeneous microtubules undergo dynamic instability, but they polymerize slower and have fewer catastrophes. Interestingly, we find that epitaxial growth of α1A/βIII microtubules from heterogeneous brain seeds is inefficient but can be fully rescued by incorporating as little as 5% of brain tubulin into the homogeneous α1A/βIII lattice. Our study establishes a system to examine the structure and dynamics of mammalian microtubules with well defined tubulin species and is a first and necessary step toward uncovering how tubulin genetic and chemical diversity is exploited to modulate intrinsic microtubule dynamics.

Cdk5 regulates developmental remodeling of mushroom body neurons in Drosophila.

During metamorphosis, axons and dendrites of the mushroom body (MB) in the Drosophila central brain are remodeled extensively to support the transition from larval to adult behaviors. We show here that the neuronal cyclin-dependent kinase, Cdk5, regulates the timing and rate of mushroom body remodeling: reduced Cdk5 activity causes a delay in pruning of MB neurites, while hyperactivation accelerates it. We further show that Cdk5 cooperates with the ubiquitin-proteasome system in this process. Finally, we show that Cdk5 modulates the first overt step in neurite disassembly, dissolution of the neuronal tubulin cytoskeleton, and provide evidence that it also acts at additional steps of MB pruning. These data show that Cdk5 regulates the onset and extent of remodeling of the Drosophila MB. Given the wide phylogenetic conservation of Cdk5, we suggest that it is likely to play a role in developmental remodeling in other systems, as well. Moreover, we speculate that the well-established role of Cdk5 in neurodegeneration may involve some of the same cellular mechanisms that it uses during developmental remodeling.

N-Acetyl-D-Glucosamine Kinase Promotes the Axonal Growth of Developing Neurons.

N-acetyl-D-glucosamine kinase (NAGK) plays an enzyme activity-independent, non-canonical role in the dendritogenesis of hippocampal neurons in culture. In this study, we investigated its role in axonal development. We found NAGK was distributed throughout neurons until developmental stage 3 (axonal outgrowth), and that its axonal expression remarkably decreased during stage 4 (dendritic outgrowth) and became negligible in stage 5 (mature). Immunocytochemistry (ICC) showed colocalization of NAGK with tubulin in hippocampal neurons and with Golgi in somata, dendrites, and nascent axons. A proximity ligation assay (PLA) for NAGK and Golgi marker protein followed by ICC for tubulin or dynein light chain roadblock type 1 (DYNLRB1) in stage 3 neurons showed NAGK-Golgi complex colocalized with DYNLRB1 at the tips of microtubule (MT) fibers in axonal growth cones and in somatodendritic areas. PLAs for NAGK-dynein combined with tubulin or Golgi ICC showed similar signal patterns, indicating a three way interaction between NAGK, dynein, and Golgi in growing axons. In addition, overexpression of the NAGK gene and of kinase mutant NAGK genes increased axonal lengths, and knockdown of NAGK by small hairpin (sh) RNA reduced axonal lengths; suggesting a structural role for NAGK in axonal growth. Finally, transfection of ‘DYNLRB1 (74–96)’, a small peptide derived from DYNLRB1’s C-terminal, which binds with NAGK, resulted in neurons with shorter axons in culture. The authors suggest a NAGK-dynein-Golgi tripartite interaction in growing axons is instrumental during early axonal development.

Microtubule Alterations Occur Early in Experimental Parkinsonism and The Microtubule Stabilizer Epothilone D Is Neuroprotective

The role of microtubule (MT) dysfunction in Parkinson's disease is emerging. It is still unknown whether it is a cause or a consequence of neurodegeneration. Our objective was to assess whether alterations of MT stability precede or follow axonal transport impairment and neurite degeneration in experimental parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in C57Bl mice. MPTP induced a time- and dose-dependent increase in fibres with altered mitochondria distribution, and early changes in cytoskeletal proteins and MT stability. Indeed, we observed significant increases in neuron-specific βIII tubulin and enrichment of deTyr tubulin in dopaminergic neurons. Finally, we showed that repeated daily administrations of the MT stabilizer Epothilone D rescued MT defects and attenuated nigrostriatal degeneration induced by MPTP. These data suggest that alteration of ΜΤs is an early event specifically associated with dopaminergic neuron degeneration. Pharmacological stabilization of MTs may be a viable strategy for the management of parkinsonism.

Transglutaminase and Polyamination of Tubulin: Posttranslational Modification for Stabilizing Axonal Microtubules

Neuronal microtubules support intracellular transport, facilitate axon growth, and form a basis for neuronal morphology. While microtubules in nonneuronal cells are depolymerized by cold, Ca(2+), or antimitotic drugs, neuronal microtubules are unusually stable. Such stability is important for normal axon growth and maintenance, while hyperstability may compromise neuronal function in aging and degeneration. Though mechanisms for stability are unclear, studies suggest that stable microtubules contain biochemically distinct tubulins that are more basic than conventional tubulins. Transglutaminase-catalyzed posttranslational incorporation of polyamines is one of the few modifications of intracellular proteins that add positive charges. Here weshow that neuronal tubulin can be polyaminated by transglutaminase. Endogenous brain transglutaminase-catalyzed polyaminated tubulins have thebiochemical characteristics of neuronal stable microtubules. Inhibiting polyamine synthesis or transglutaminase activity significantly decreases microtubule stability invitro and invivo. Together, these findings suggest that transglutaminase-catalyzed polyamination of tubulins stabilizes microtubules essential for unique neuronal structures and functions.