Microtubule-associated Protein 1A Research Articles

Phosphorylation of MAP 1A regulates hyperphosphorylation of Tau in Alzheimer's disease model.

Hyperphosphorylation of Tau is one of the important pathological features of Alzheimer's disease (AD). Therefore, studying the mechanisms behind Tau hyperphosphorylation is crucial in exploring the pathogenesis of neurological damage in AD. In this study, after the establishment of rat models of AD, quantitative phosphoproteomics and proteomics were performed to identify proteins, showing that phosphorylation of microtubule associated protein 1A (MAP 1A) was lower in the model group. Western blot confirmed the changes of MAP 1A in the SD rats, APP/PS1 transgenic mice and cell AD models. To further study the molecular mechanism of recombinant MAP 1A phosphorylation affecting Tau phosphorylation, interfering siRNA-MAP 1A and protein immunoprecipitation reaction analysis were performed in AD cell models. Cyclin-dependent kinase 5 (CDK5) showed reduced binding to MAP 1A and increased binding to Tau, resulting in a decrease in phosphorylated MAP 1A (p-MAP 1A) and an increase in phosphorylated Tau (p-Tau), and MAP 1A silencing promoted binding of CDK5-Tau and increased Tau phosphorylation, thereby reducing the cell survival rate. In summary, we found that p-MAP 1A downregulation associated with p-Tau upregulation was due to their altered binding forces to CDK5, and MAP 1A could enhance autophosphorylation by competitive binding to CDK5 and antagonise Tau phosphorylation. This leads to neuronal protection and reducing tissue damage levels in AD, which can help better understand the mechanisms of AD pathogenesis.

Intracellular Accumulation of α-Synuclein Aggregates Promotes S-Nitrosylation of MAP1A Leading to Decreased NMDAR-Evoked Calcium Influx and Loss of Mature Synaptic Spines.

Cortical synucleinopathies, including dementia with Lewy bodies and Parkinson's disease dementia, collectively known as Lewy body dementia, are characterized by the aberrant aggregation of misfolded α-synuclein (α-syn) protein into large inclusions in cortical tissue, leading to impairments in proteostasis and synaptic connectivity and eventually resulting in neurodegeneration. Here, we show that male and female rat cortical neurons exposed to exogenous α-syn preformed fibrils accumulate large, detergent-insoluble, PS129-labeled deposits at synaptic terminals. Live-cell imaging of calcium dynamics coupled with assessment of network activity reveals that aberrant intracellular accumulation of α-syn inhibits synaptic response to glutamate through NMDARs, although deficits manifest slowly over a 7 d period. Impairments in NMDAR activity temporally correlated with increased nitric oxide synthesis and S-nitrosylation of the dendritic scaffold protein, microtubule-associated protein 1A. Inhibition of nitric oxide synthesis via the nitric oxide synthase inhibitor l-NG-nitroarginine methyl ester blocked microtubule-associated protein 1A S-nitrosylation and normalized NMDAR-dependent inward calcium transients and overall network activity. Collectively, these data suggest that loss of synaptic function in Lewy body dementia may result from synucleinopathy-evoked nitrosative stress and subsequent NMDAR dysfunction.SIGNIFICANCE STATEMENT This work shows the importance of the redox state of microtubule-associated protein 1A in the maintenance of synaptic function through regulation of NMDAR. We show that α-syn preformed fibrils promote nitric oxide synthesis, which triggers S-nitrosylation of microtubule-associated protein 1A, leading to impairment of NMDAR-dependent glutamate responses. This offers insight into the mechanism of synaptic dysfunction in Lewy body dementia.

CK1 Is a Druggable Regulator of Microtubule Dynamics and Microtubule-Associated Processes.

Simple SummaryThe Casein Kinase 1 (CK1) family of serine-threonine specific protein kinases regulates the activity of key regulatory proteins and signaling pathways being involved in embryonic development but also in the adult organism. Furthermore, it plays an important role in the regulation of proliferation, differentiation, apoptotic processes, circadian rhythm, chromosome segregation, and other microtubule-associated processes. Deregulation of CK1 expression and activity, as well as mutations in the coding region, contribute to the development of many human pathologies, including cancer. Alternations in the site-specific phosphorylation of α/β-tubulin and microtubule-associated proteins affect microtubule stability, finally resulting in mitotic defects and genomic instability. Here we review our knowledge about CK1 functions in general and especially in chromosome segregation. Furthermore, an update in modulating CK1 activity by small molecule inhibitors and peptides specifically inhibiting CK1 protein interactions as new therapy concepts for the treatment of cancer will be discussed.Protein kinases of the Casein Kinase 1 family play a vital role in the regulation of numerous cellular processes. Apart from functions associated with regulation of proliferation, differentiation, or apoptosis, localization of several Casein Kinase 1 isoforms to the centrosome and microtubule asters also implicates regulatory functions in microtubule dynamic processes. Being localized to the spindle apparatus during mitosis Casein Kinase 1 directly modulates microtubule dynamics by phosphorylation of tubulin isoforms. Additionally, site-specific phosphorylation of microtubule-associated proteins can be related to the maintenance of genomic stability but also microtubule stabilization/destabilization, e.g., by hyper-phosphorylation of microtubule-associated protein 1A and RITA. Consequently, approaches interfering with Casein Kinase 1-mediated microtubule-specific functions might be exploited as therapeutic strategies for the treatment of cancer. Currently pursued strategies include the development of Casein Kinase 1 isoform-specific small molecule inhibitors and therapeutically useful peptides specifically inhibiting kinase-substrate interactions.

Changes in the Number and Morphology of Dendritic Spines in the Hippocampus and Prefrontal Cortex of the C58/J Mouse Model of Autism

Autism spectrum disorder (ASD) has a broad range of neurobiological characteristics, including alterations in dendritic spines, where approximately 90% of excitatory synapses occur. Therefore, changes in their number or morphology would be related to atypical brain communication. The C58/J inbred mouse strain displays low sociability, impaired communication, and stereotyped behavior; hence, it is considered among the animal models suitable for the study of idiopathic autism. Thus, this study aimed to evaluate the dendritic spine differences in the hippocampus and the prefrontal cortex of C58/J mice. We found changes in the number of spines and morphology in a brain region-dependent manner: a subtle decrease in spine density in the prefrontal cortex, higher frequency of immature phenotype spines characterized by filopodia-like length or small morphology, and a lower number of mature phenotype spines with mushroom-like or wide heads in the hippocampus. Moreover, an in silico analysis showed single nucleotide polymorphisms (SNPs) at genes collectively involved in regulating structural plasticity with a likely association with ASD, including MAP1A (Microtubule-Associated Protein 1A), GRM7 (Metabotropic Glutamate Receptor, 7), ANKRD11 (Ankyrin Repeat Domain 11), and SLC6A4 (Solute Carrier Family 6, member 4), which might support the relationship between the C58/J strain genome, an autistic-like behavior, and the observed anomalies in the dendritic spines.

Normobaric Oxygen (NBO) Therapy Reduces Cerebral Ischemia/Reperfusion Injury through Inhibition of Early Autophagy.

Objectives Normobaric oxygen (NBO) therapy has great clinical potential in the treatment of ischemic stroke, but its underlying mechanism is unknown. Our study aimed to investigate the role of autophagy during the application of NBO on cerebral ischemia/reperfusion injury. Methods Male Sprague Dawley rats received 2 hours of middle cerebral artery occlusion (MCAO), followed by 2, 6, or 24 hours of reperfusion. At the beginning of reperfusion, rats were randomly given NBO (95% O2) or room air (21% O2) for 2 hours. In some animals, 3-methyladenine (3-MA, autophagy inhibitor) was administered 10 minutes before reperfusion. The severity of the ischemic injury was determined by infarct volume, neurological deficit, and apoptotic cell death. Western blotting was used to determine the protein expression of autophagy and apoptosis, while mRNA expression of apoptotic molecules was detected by real-time PCR. Results NBO treatment after ischemia/reperfusion significantly decreased infarct volume and neurobehavioral defects. The increased expression of the autophagy markers, including microtubule-associated protein 1A light chain 3 (LC3) and Beclin 1, after ischemia/reperfusion was reversed by NBO, while promoting Sequestosome 1 (p62/SQSTM1) expression. In addition, NBO reduced cerebral apoptosis in association with alleviated BAX expression and increased BCL-2 expression. 3-MA reduced autophagy and apoptotic death but did not further improve NBO-attenuated ischemic damage. Conclusion NBO induced remarkable neuroprotection from ischemic injury, which was correlated with blocked autophagy activity.

Tau knockout exacerbates degeneration of parvalbumin-positive neurons in substantia nigra pars reticulata in Parkinson's disease-related α-synuclein A53T mice.

α-Synuclein (α-syn)-induced neurotoxicity has been generally accepted as a key step in the pathogenesis of Parkinson's disease (PD). Microtubule-associated protein tau, which is considered second only to α-syn, has been repeatedly linked with PD in association studies. However, the underlying interaction between these two PD-related proteins in vivo remains unclear. To investigate how the expression of tau affects α-syn-induced neurodegeneration in vivo, we generated triple transgenic mice that overexpressed α-syn A53T mutation in the midbrain dopaminergic neurons (mDANs) with different expression levels of tau. Here, we found that tau had no significant effect on the A53T α-syn-mediated mDANs degeneration. However, tau knockout could modestly promote the formation of α-syn aggregates, accelerate the severe and progressive degeneration of parvalbumin-positive (PV+) neurons in substantia nigra pars reticulata (SNR), accompanied with anxiety-like behavior in aged PD-related α-syn A53T mice. The mechanisms may be associated with A53T α-syn-mediated specifically successive impairment of N-methyl-d-aspartate receptor subunit 2B (NR2B), postsynaptic density-95 (PSD-95) and microtubule-associated protein 1A (MAP1A) in PV+ neurons. Our study indicates that MAP1A may play a beneficial role in preserving the survival of PV+ neurons, and that inhibition of the impairment of NR2B/PSD-95/MAP1A pathway, may be a novel and preferential option to ameliorate α-syn-induced neurodegeneration.

Autism spectrum disorder and attention deficit hyperactivity disorder have a similar burden of rare protein-truncating variants.

We analyze the exome sequences of approximately 8,000 children with autism spectrum disorder (ASD) and/or attention-deficit/hyperactivity disorder (ADHD) and 5,000 controls, and we find that ASD and ADHD have a similar burden of rare protein-truncating variants in evolutionarily constrained genes, both significantly higher than controls. This motivates a combined analysis across ASD and ADHD, which identifies microtubule-associated protein 1A (MAP1A) as a novel exome-wide significant gene conferring risk for childhood psychiatric disorders.

Protective effects of extracellular polymeric substances from Aphanizomenon flos-aquae on neurotoxicity induced by local anesthetics.

The neurotoxicity of local anesthetics has received an increasing amount of attention and more effective therapeutic agents are required. Extracellular polymeric substances from Aphanizomenon flos-aquae (EPS-A) are high molecular weight polysaccharides. The present study aimed to elucidate the protective effects of EPS-A on neurotoxicity induced by local anesthetics in an intraperitoneal injection bupivacaine rat model. The results of immunohistochemical staining inicated that following intraperitoneal injection of EPS-A the levels of apoptosis and caspase-3 decreased, and the expression levels of microtubule-associated protein 1A light chain 3 (LC3) and beclin1 increased. In order to further clarify the mechanism of the EPS-A-mediated protection, the expression of key proteins associated with autophagy was investigated by western blotting. The results suggested that the ratio of LC3-II/LC3-I and the expression level of beclin1 increased. Taken together, the results indicated that EPS-A induced neuroprotective effects on bupivacaine-induced neurotoxicity. The underlying mechanism may be associated with the inhibition of apoptosis, upregulation of autophagy and improvement of cell survival. The results suggested that EPS-A may be a candidate neuroprotective agent against neurotoxicity caused by local anesthetics.

Structure of the PSD-95/MAP1A complex reveals a unique target recognition mode of the MAGUK GK domain.

The PSD-95 family of membrane-associated guanylate kinases (MAGUKs) are major synaptic scaffold proteins and play crucial roles in the dynamic regulation of dendritic remodelling, which is understood to be the foundation of synaptogenesis and synaptic plasticity. The guanylate kinase (GK) domain of MAGUK family proteins functions as a phosphor-peptide binding module. However, the GK domain of PSD-95 has been found to directly bind to a peptide sequence within the C-terminal region of neuronal-specific microtubule-associated protein 1A (MAP1A), although the detailed molecular mechanism governing this phosphorylation-independent interaction at the atomic level is missing. In the present study, we determine the crystal structure of PSD-95 GK in complex with the MAP1A peptide at 2.6-Å resolution. The complex structure reveals that, unlike a linear and elongated conformation in the phosphor-peptide/GK complexes, the MAP1A peptide adopts a unique conformation with a stretch of hydrophobic residues far from each other in the primary sequence clustering and interacting with the 'hydrophobic site' of PSD-95 GK and a highly conserved aspartic acid of MAP1A (D2117) mimicking the phosphor-serine/threonine in binding to the 'phosphor-site' of PSD-95 GK. We demonstrate that the MAP1A peptide may undergo a conformational transition upon binding to PSD-95 GK. Further structural comparison of known DLG GK-mediated complexes reveals the target recognition specificity and versatility of DLG GKs.

Defects in Synaptic Plasticity, Reduced NMDA-Receptor Transport, and Instability of Postsynaptic Density Proteins in Mice Lacking Microtubule-Associated Protein 1A.

This work is the first report showing the significance of non-motor microtubule-associated protein in maintaining synaptic plasticity thorough a novel mechanism: anchoring of NMDA receptors to cytoskeleton supports transport of NMDA receptors and stabilizes postsynaptic density scaffolds binding to NMDA receptors. Newly generated mutant mice lacking MAP1A exhibited learning disabilities and reduced synaptic plasticity attributable to disruptions of the anchoring machinery.

Mutations in the microtubule-associated protein 1A (Map1a) gene cause Purkinje cell degeneration.

The structural microtubule-associated proteins (MAPs) are critical for the organization of neuronal microtubules (MTs). Microtubule-associated protein 1A (MAP1A) is one of the most abundantly expressed MAPs in the mammalian brain. However, its in vivo function remains largely unknown. Here we describe a spontaneous mouse mutation, nm2719, which causes tremors, ataxia, and loss of cerebellar Purkinje neurons in aged homozygous mice. The nm2719 mutation disrupts the Map1a gene. We show that targeted deletion of mouse Map1a gene leads to similar neurodegenerative defects. Before neuron death, Map1a mutant Purkinje cells exhibited abnormal focal swellings of dendritic shafts and disruptions in axon initial segment (AIS) morphology. Furthermore, the MT network was reduced in the somatodendritic and AIS compartments, and both the heavy and light chains of MAP1B, another brain-enriched MAP, was aberrantly distributed in the soma and dendrites of mutant Purkinje cells. MAP1A has been reported to bind to the membrane-associated guanylate kinase (MAGUK) scaffolding proteins, as well as to MTs. Indeed, PSD-93, the MAGUK specifically enriched in Purkinje cells, was reduced in Map1a(-/-) Purkinje cells. These results demonstrate that MAP1A functions to maintain both the neuronal MT network and the level of PSD-93 in neurons of the mammalian brain.

Highly expressed genes in human high grade gliomas: immunohistochemical analysis of data from the human protein atlas.

Gene expression within human glioblastomas were analyzed from data on 20,083 genes entered into the on-line Human Protein Atlas. In selecting genes that are strongly expressed within normal human brain tissue, 58 genes were identified from a search of the 20,083 entries that were rated as showing 90% or greater intensity of expression within normal brain tissues. Of these 58, a subset of 48 genes was identified that not only had expression data for human glioblastomas but also for the human glioblastoma cell line U-251. Four of these 48 selected genes were found to be strongly expressed within the cytoplasm when assessed by both histologic sampling of high grade glioma patient cases as well as U-251 glioblastoma cell line immunofluoresence analysis. These four human genes are: AGBL2 (ATP/GTP binding protein-like 2), BLOC1S6 (biogenesis of lysosomal organelles complex-1, subunit 6), MAP1A (microtubule-associated protein 1A) and ZSWIM5 (zinc finger, SWIM-type containing 5, also known as KIAA1511). Further research is advocated to investigate the role of ZSWIM5 and AGBL2 in glioma cell biology.

Downregulated adaptor protein p66Shc mitigates autophagy process by low nutrient and enhances apoptotic resistance in human lung adenocarcinoma A549 cells

Macroautophagy or autophagy is a lysosome-dependent process in which enzymatic degradation and recycling of cytosolic components occur in stressful contexts. The mechanisms underlying the signaling from starvation to the regulation of autophagy are not fully understood. We previously showed that the Src family member p66(Shc) (focal adhesion-associated 66 kDa isoform of the Src homology and collagen) promotes anoikis and suppresses tumor metastasis via k-Ras-dependent control of proliferation and survival. However, the role of p66(Shc) in low-nutrient-induced autophagy-related pathways remains elusive. In this work, human lung adenocarcinoma A549 cells were used to further investigate the biological effects of p66(Shc) on autophagy and apoptotic resistance. Here, we show that deficiency of p66(Shc) mitigates the low-nutrient-induced autophagy process in the levels of microtubule-associated protein 1A light chain protein 3B (LC3B) conversion, in the number of autophagic vacuoles and in p62/sequestosome 1 protein degradation. However, autophagy-related protein Beclin 1 was not significantly changed during low-nutrient treatment. Furthermore, we found that prolonged phosphorylation of extracellular signaling-regulated kinase (Erk)1/2, but not phosphorylation of Akt is significantly sustained when p66(Shc) expression is inhibited by shRNA. In addition, cleavage of caspase 7 and poly(ADP-ribose) polymerase, but not caspase 6 and 9 are retarded with this effect compared to the shRNA control cells. Together, these findings suggest the possibility that p66(Shc) plays a pivotal role in coordinately regulating autophagy process and apoptotic resistance in A549 cells under nutrient-limited conditions.

Physical exercise improves functional recovery through mitigation of autophagy, attenuation of apoptosis and enhancement of neurogenesis after MCAO in rats

BackgroundPhysical exercise improves functional recovery after stroke through a complex mechanism that is not fully understood. Transient focal cerebral ischemia induces autophagy, apoptosis and neurogenesis in the peri-infarct region. This study is aimed to examine the effects of physical exercise on autophagy, apoptosis and neurogenesis in the peri-infarct region in a rat model of transient middle cerebral artery occlusion (MCAO).ResultsWe found that autophagosomes, as labeled by microtubule-associated protein 1A light chain 3-II (LC3-II), were evident in the peri-infarct region at 3 days after 90-minute MCAO. Moreover, 44.6% of LC3-positive cells were also stained with TUNEL. The number of LC3 positive cells was significantly lower in physical exercise group than in control group at 14 and 21 days after MCAO. Suppression of autophagosomes by physical exercise was positively associated with improvement of neurological function. In addition, physical exercise significantly decreased the number of TUNEL-positive cells and increased the numbers of Ki67-positive, a proliferative marker, and insulin-like growth factor-1 (IGF-1) positive cells at 7, 14, and 21 days after MCAO.ConclusionsThe present results demonstrate that physical exercise enhances neurological function possibly by reduction of autophagosome accumulation, attenuation of apoptosis and enhancement of neurogenesis in the peri-infarct region after transient MCAO in rats.

The Microtubule-Associated Protein 1A (MAP1A) is an Early Molecular Target of Soluble Aβ-Peptide

A progressive accumulation of amyloid β-protein (Aβ) is widely recognized as a pathological hallmark of Alzheimer's disease (AD). Substantial progress has been made toward understanding the neurodegenerative cascade initiated by small soluble species of Aβ and recent evidence supports the notion that microtubule rearrangements may be proximate to neuritic degeneration and deficits in episodic declarative memory. Here, we examined primary cortical neurons for changes in markers associated with synaptic function following exposure to sublethal concentrations of non-aggregated Aβ-peptide. This data show that soluble Aβ species at a sublethal concentration induce degradation of the microtubule-associated protein 1A (MAP1A) without concurrently affecting dendritic marker MAP2 and/or the pre-synaptic marker synaptophysin. In addition, MAP1A was found to highly co-localize with the postsynaptic density-95 (PSD-95) protein, proposing that microtubule perturbations might be central for the Aβ-induced neuronal dysfunctions as PSD-95 plays a key role in synaptic plasticity. In conclusion, this study suggests that disruption of MAP1A could be a very early manifestation of Aβ-mediated synaptic dysfunction-one that presages the clinical onset of AD by years. Moreover, our data support the notion of microtubule-stabilizing agents as effective AD drugs.

Autophagosomes accumulation is associated with β‐amyloid deposits and secondary damage in the thalamus after focal cortical infarction in hypertensive rats

Focal cerebral cortical infarction after distal middle cerebral artery occlusion causes β-amyloid deposition and secondary neuronal degeneration in the ipsilateral ventroposterior nucleus of the thalamus. Several studies suggest that autophagy is an active pathway for β-amyloid peptide generation. This study aimed to investigate the role of autophagy in thalamic β-amyloid deposition and neuronal degeneration after cerebral cortical infarction in hypertensive rats. At 7 and 14days after middle cerebral artery occlusion, neuronal death and β-amyloid deposits were evident in the ipsilateral ventroposterior nucleus, and the activity of β-site amyloid precursor protein (APP)-cleaving enzyme 1, required for β-amyloid peptide generation, was elevated in the thalamus. In correlation, both the number of cells showing punctate microtubule-associated protein 1A light chain 3 fluorescence and levels of light chain 3-II protein, an autophagosome marker, were markedly increased. Notably, most of the cells that over-expressed β-site APP-cleaving enzyme 1 displayed punctate light chain 3 staining. Furthermore, the inhibition of autophagy with 3-methyladenine significantly reduced the thalamic neuronal damage, β-amyloid deposits, and β-site APP-cleaving enzyme 1 activity. These results suggest that autophagosomes accumulate within thalamic cells after cerebral cortical infarction, which is associated with thalamic β-amyloid deposition and secondary neuronal degeneration via elevation of β-site APP-cleaving enzyme 1 level.

Cognitive and histological disturbances after chlorpyrifos exposure and chronic Aβ(1–42) infusions in Wistar rats

Exposure to pesticides has been linked to an increased vulnerability to neurodegenerative diseases. In order to study whether the exposure to the organophosphate chlorpyrifos renders the brain prone to amyloid-beta peptide deposition and accelerates its neuropathological and behavioural effects, Wistar rats were injected a single subcutaneous dose of chlorpyrifos (250mg/kg) and subsequently infused with Aβ(1–42) peptide (i.c.v.) for 15 days. No effects of either treatment were noted in the classic water maze test. The animals infused with Aβ peptide showed worse performance when the platform was both hidden and moved from trial to trial. Both groups showed worse performance when the platform was visible and moved from trial to trial. No amyloid deposition was observed in hippocampus or cerebral cortex after the infusion period, although microtubule-associated protein 1A (MAP1A) immunoreactivity was significantly reduced in hippocampus and prefrontal cortex, whereas chlorpyrifos exposure produced a significant reduction of microtubule-associated protein 2 (MAP2) in the prefrontal cortex. Therefore, behavioural deficits could be related to a loss of dendrite and spine processes in these brain regions.

Microtubule-associated Protein 1S (MAP1S) Bridges Autophagic Components with Microtubules and Mitochondria to Affect Autophagosomal Biogenesis and Degradation

The ubiquitously distributed MAP1S is a homologue of the exclusively neuronal distributed microtubule-associated protein 1A and 1B (MAP1A/B). They give rise to multiple isoforms through similar post-translational modification. Isoforms of MAP1S have been implicated in microtubule dynamics and mitotic abnormalities and mitotic cell death. Here we show that ablation of the Map1s gene in mice caused reduction in the B-cell CLL/lymphoma 2 or xL (Bcl-2/xL) and cyclin-dependent kinase inhibitor 1B (P27) protein levels, accumulation of defective mitochondria, and severe defects in response to nutritive stress, suggesting defects in autophagosomal biogenesis and clearance. Furthermore, MAP1S isoforms interacted with the autophagosome-associated light chain 3 of MAP1A/B (LC3), a homologue of yeast autophagy-related gene 8 (ATG8), and recruited it to stable microtubules in a MAP1S and LC3 isoform-dependent mode. In addition, MAP1S interacted with mitochondrion-associated leucine-rich PPR-motif containing protein (LRPPRC) that interacts with the mitophagy initiator and Parkinson disease-related protein Parkin. The three-way interactions of MAP1S isoforms with LC3 and microtubules as well as the interaction of MAP1S with LRPPRC suggest that MAP1S isoforms may play positive roles in integration of autophagic components with microtubules and mitochondria in both autophagosomal biogenesis and degradation. For the first time, our results clarify roles of MAP1S in bridging microtubules and mitochondria with autophagic and mitophagic initiation, maturation, trafficking, and lysosomal clearance. Defects in the MAP1S-regulated autophagy may impact heart disease, cancers, neurodegenerative diseases, and a wide range of other diseases.

ENHANCEMENT OF MICROTUBULE-ASSOCIATED PROTEIN-1 ALPHA GENE EXPRESSION IN OSTEOBLASTS BY LOW LEVEL LASER IRRADIATION

Low level laser irradiation (LLLI) stimulates bone regeneration. However, the molecular mechanisms leading to this is not yet understood. The stepwise subtractive cDNA cloning technology has been developed, coupled with DNA homology searched in DNA database is useful to identify the certain gene. In order to understand the mechanism, we attempted to identify genes whose expressions are enhanced by LLLI. MC3T3-E1 osteoblastic cells were irradiated with an 830 nm Ga-Al-As diode laser, and a cDNA library was constructed using subtractive gene cloning. The cDNA library of osteoblasts which was treated by LLLI was constructed. Nucleotide sequences were analyzed and homology searched in a DNA database using BLASTN program to identify the gene with altered expression. Altered mRNA levels by LLLI were confirmed by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. The DNA sequence of a subtracted gene clone MCL129 indicated high homology (99%) with the microtubule-associated protein 1A (MAP1A) gene. Increase in MAP-1A mRNA level by LLLI was successfully confirmed by RT-PCR and real-time PCR. MAP1A has been shown to promote microtubule assembly and its functional expression. Microtubules play an important role in cell division, cell shape and polarity, cell movement, intracellular transport, signal transduction, and synthesis and secretion of collagen. Thus, enhancement of MAP1A gene expression by LLLI may be one of the molecular mechanisms involved in accelerating bone formation by LLLI. LLLI irradiation enhances MAP1A gene expression and modulates microtubule assembly and the functional structure of microtubules, in turn, stimulates osteoblastic proliferation and differentiation.

Phosphoproteomic Analysis Reveals Site-Specific Changes in GFAP and NDRG2 Phosphorylation in Frontotemporal Lobar Degeneration

Frontotemporal lobar degeneration (FTLD) is a progressive neurodegenerative disease characterized by behavioral abnormalities, personality changes, language dysfunction, and can co-occur with the development of motor neuron disease. One major pathological form of FTLD is characterized by intracellular deposition of ubiquitinated and phosphorylated TAR DNA binding protein-43 (TDP-43), suggesting that dysregulation in phosphorylation events may contribute to disease progression. However, to date systematic analysis of the phosphoproteome in FTLD brains has not been reported. In this study, we employed immobilized metal affinity chromatography (IMAC) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify phosphopeptides from FTLD and age-matched control post-mortem human brain tissue. Using this approach, we identified 786 phosphopeptides in frontal cortex (control and FTLD), in which the population of phosphopeptides represented approximately 50% of the total peptides analyzed. Label-free quantification using spectral counts revealed six proteins with significant changes in the FTLD phosphoproteome. N-myc-Downstream regulated gene 2 (NDRG2) and glial fibrillary acidic protein (GFAP) had an increased number of phosphospectra in FTLD, whereas microtubule associated protein 1A (MAP1A), reticulon 4 (RTN4; also referred to as neurite outgrowth inhibitor (Nogo)), protein kinase C gamma (PRKCG), and heat shock protein 90 kDa alpha, class A member 1(HSP90AA1) had significantly fewer phosphospectra compared to control brain. To validate these differences, we examined NDRG2 phosphorylation in FTLD brain by immunoblot analyses, and using a phosphoserine-13 (pSer13) GFAP monoclonal antibody we show an increase in pSer13 GFAP levels by immunoblot concomitant with increased overall GFAP levels in FTLD cases. These data highlight the utility of combining proteomic and phosphoproteomic strategies to characterize post-mortem human brain tissue.