In our previous studies it has been demonstrated that phosphorylation of the neuron specific intermediate filament proteins, such as neurofilaments (NFs) were tightly regulated developmentally and topographically, and generally confined to the axonal compartment and selectively occurs on proline directed serine (Ser) and threonine (Thr) residues. It was recognized that in neurodegenerative disorders such as Alzheimers disease (AD) and Amyotrophic lateral sclerosis (ALS), the pathology was characterized by an accumulation of aberrantly phosphorylated cytoskeletal proteins in perikarya on these residues, suggesting that topographic regulation had been compromised. Neurodegenerative diseases including AD are complex and chronic disorders that involve the disruption of the neuronal network in the human brain. We have made first time the comprehensive study of quantitative mass spectrometric analysis of human neuron specific major cytoskeletal proteins, medium (NF-M)and high (NF-H) molecular weight neurofilament proteins from control adult and AD matched brains. This study aimed to extend our knowledge of specific phosphorylation sites to provide insight into the role of kinases and phosphatases in the formation of neurofibrillary tangles (NFTs) and aggregates. The quantitative mass spectroscopic technique ( iTRAQ) used in this study identified specific phosphorylation sites among multiple lysine serine/threonine proline (KS/TP) repeats and non-S/TP sites in NF-M/H. For example, evidence of phosphorylation of proline-directed and non-proline-directed Ser residue suggests the involvement of both proline-directed and non-proline-directed kinases in AD.The iTRAQ analysis revealed primarily that specific Ser/Thr sites are phosphorylated at a higher fold in AD brain compared with control brain and that not all the neurofilament KSP sites are phosphorylated at similar levels in AD. Some of them are phosphorylated at a greater abundance compared with other sites. Hipocampal,CA1 pyramidal cells are known for susceptibility to neurofibrillary degeneration in AD. In human brain, these neurons also show an age-related perikaryal accumulation of NFs. Most of the phosphorylation sites in AD tangles are perikaryonal constituents. Normal perikarya do not contain phosphorylated neurofilament proteins.Our immunohistochemistry data suggest that phospho-NF is predominantly detected in intracellular NFTs and extracellular plaques in AD. The iTRAQ data obtained with phosphorylated NF-M from AD brain also show the higher abundance of phosphorylation of non-proline-directed Ser or Thr residues,suggesting the involvement of also non-proline-directed kinases in addition to proline - directed kinases in AD brain. This study first time demonstrates the importance of regulation of NF-M phosphorylation in physiology and pathology. It is found that KSP repeat phosphorylation of NF-M is significantly higher compared with NF-H, despite the greater number of these sites in NF-H. We were able to obtain the sequence coverage for 13/18 phosphorylation sites of NF-M;however, iTRAQ analysis resulted in the aberrant phosphorylation of 10/43 phosphorylation sites of NF-H in AD brain. Based on the stoichiometry, there is 2-fold higher KSP repeats phosphorylated in NF-M compared with NF-H;therefore, we believe that NF-M might contribute more to aberrant phosphorylation in AD compared with other cytoskeletal proteins and also suggests that specific antibodies could be generated to the SP and non-SP sites that are phosphorylated at a greater abundance and can be used as bio-markers in AD and the specific inhibitors of these kinase can be used as therapeutic reagents for AD and other neurodegenerative diseases involved in aberrant neuronal cyytoskeletal protein phosphorylation. NFTs are predominant feature in the pyramidal cells of hippocampus and in the cerebral cortex of the people suffering from AD. Tau deposits occur in dystrophic neuritis, as fine neuropil threads, and as massive neurofibrillary tangles in neuronal cell bodies. Do NFTs contain proteins other than tau? We have convincingly demonstrated by an independent method, i.e., mass spectrometry and immunohistochemistry, that NFs, NF-L, NF-M and NF-H, are indeed, integral components of NFTs in AD brains. In addition to these NF phosphopeptides (NF-M, NF-H), three non-phosphopeptides have also been recorded, including two from NF-L. The presence of all three NF subunits suggests that intact 10 nm NFs may also be integrated into PHF/NFTs. The mass spectroscopic and immunochemical data presented in this study confirm a series of immunological studies in the eighties which demonstrated NFs in NFTs in situ and in preparations of PHF purified from AD brain tissue. Western blots of purified PHF preparations with NF phospho-specific antibodies, SMI31 and RT97 also showed robust expression of NF-H at 250 kDa and NF-M at 150 kDa.Although NFs (NFs) have been shown immunohistologically to be part of NFTs, there has been debate that the identity of NF proteins in NFTs is due to the cross-reactivity of phosphorylated NF-antibodies with phospho-Tau. We have provided direct evidence on the identity of NFs in NFTs by immunochemical and mass spectrometric analysis. We have purified sarkosyl-insoluble NFTs and performed liquid chromatography (LC)/mass spectrometry MS/MS of NFT tryptic digests. The phosphoproteomics of NFTs clearly identified an NF-M phosphopeptides, SPVPKS*PVEEAK corresponding to Ser685, KAES*PVKEEAVAEVVTITK corresponding to Ser736 and NF-H phosphopeptide, EPDDAKAKEPS*KP corresponding to Ser942. Western blotting of purified tangles with SMI31 showed a 150 kDa band corresponding to phospho-NF-M while RT97 antibodies detected phospho-NF-H. The proteomics analysis also identified an NF-L peptide (ALYEQEIR, EAEEEKKVEGAGEEQAAAK) and another intermediate filament protein, vimentin (FADLSEAANR). Mass spectrometry revealed Tau phosphopeptides corresponding to Thr231, Ser235, Thr-181, Ser184, Ser185, Thr212, Thr-217, Ser396, and Ser403. And finally, phosphopeptides corresponding to MAP1B corresponding to Ser1270, Ser1274, Ser1779 and MAP2 corresponding to Thr350, Ser1702 and Ser1706 were identified. In corresponding matched control preparations of PHF/NFTs, none of these phosphorylated neuronal cytoskeletal proteins were found. These studies independently demonstrate for the first time that NF proteins are integral part of NFTs in AD.

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