Neurofilament Protein Subunits Research Articles

Regulation of neurofilament length and transport by a dynamic cycle of phospho-dependent polymer severing and annealing.

Neurofilaments are cargoes of axonal transport which are unique among known intracellular cargoes in that they are long, flexible protein polymers. These polymers are transported into axons, where they accumulate in large numbers to drive the expansion of axon caliber, which is an important determinant of axonal conduction velocity. We reported previously that neurofilaments can be lengthened by joining ends, called end-to-end annealing, and that they can be shortened by severing. Here, we show that neurofilament annealing and severing are robust and quantifiable phenomena in cultured neurons that act antagonistically to regulate neurofilament length. We show that this in turn regulates neurofilament transport and that severing is regulated by N-terminal phosphorylation of the neurofilament subunit proteins. We propose that focal destabilization of intermediate filaments by site-directed phosphorylation may be a general enzymatic mechanism for severing these cytoskeletal polymers, providing a mechanism to regulate the transport and accumulation of neurofilaments in axons.

Autophagy is a novel pathway for neurofilament protein degradation in vivo

ABSTRACT How macroautophagy/autophagy influences neurofilament (NF) proteins in neurons, a frequent target in neurodegenerative diseases and injury, is not known. NFs in axons have exceptionally long half-lives in vivo enabling formation of large stable supporting networks, but they can be rapidly degraded during Wallerian degeneration initiated by a limited calpain cleavage. Here, we identify autophagy as a previously unrecognized pathway for NF subunit protein degradation that modulates constitutive and inducible NF turnover in vivo. Levels of NEFL/NF-L, NEFM/NF-M, and NEFH/NF-H subunits rise substantially in neuroblastoma (N2a) cells after blocking autophagy either with the phosphatidylinositol 3-kinase (PtdIns3K) inhibitor 3-methyladenine (3-MA), by depleting ATG5 expression with shRNA, or by using both treatments. In contrast, activating autophagy with rapamycin significantly lowers NF levels in N2a cells. In the mouse brain, NF subunit levels increase in vivo after intracerebroventricular infusion of 3-MA. Furthermore, using tomographic confocal microscopy, immunoelectron microscopy, and biochemical fractionation, we demonstrate the presence of NF proteins intra-lumenally within autophagosomes (APs), autolysosomes (ALs), and lysosomes (LYs). Our findings establish a prominent role for autophagy in NF proteolysis. Autophagy may regulate axon cytoskeleton size and responses of the NF cytoskeleton to injury and disease.

Achyranthes bidentata polypeptide k enhances the survival, growth and axonal regeneration of spinal cord motor neurons in vitro.

Achyranthes bidentata polypeptide k (ABPPk), a powerful active component from a traditional Chinese medicinal herb-Achyranthes bidentata Bl., has exhibited promising neuroprotective activity due to its multiple-targeting capability. However, the effect of ABPPk on the survival, growth and axonal regeneration of spinal cord motor neurons remains unclear. Here, a modified method, which is more optimized for embryonic cells in ambient carbon dioxide levels, was used for acquisition of rat embryonic spinal cord motor neurons with high survival and purity. ABPPk concentration-dependently enhanced the neuronal viability and promoted the neurite outgrowth. Co-culture of motor neurons and skeletal myocytes model indicated that ABPPk enhanced the neuromuscular junction development and maturation. A microfluidic axotomy model was further established for the axonal disconnection, and ABPPk significantly accelerated the axonal regeneration of motor neurons. Furthermore, we demonstrated that the upregulation of three neurofilament protein subunits in motor neurons might be relevant to the mechanisms of the growth-promoting effect of ABPPk. Our findings provide an experimental and theoretical basis for the development of ABPPk as a potential application in the development of treatment strategy for nerve injury diseases.

Charcot-Marie-Tooth disease Type 2E/1F mutant neurofilament proteins assemble into neurofilaments.

Charcot-Marie-Tooth disease Type 2E/1F (CMT2E/1F) is a peripheral neuropathy caused by mutations in neurofilament protein L (NFL), which is one of five neurofilament subunit proteins that co-assemble to form neurofilaments in vivo. Prior studies on cultured cells have shown that CMT2E/1F mutations disrupt neurofilament assembly and lead to protein aggregation, suggesting a possible disease mechanism. However, electron microscopy of axons in peripheral nerve biopsies from patients has revealed accumulations of neurofilament polymers of normal appearance and no evidence of protein aggregates. To reconcile these observations, we reexamined the assembly of seven CMT2E/1F NFL mutants in cultured cells. None of the mutants assembled into homopolymers in SW13vim- cells, but P8R, P22S, L268/269P, and P440/441L mutant NFL assembled into heteropolymers in the presence of neurofilament protein M (NFM) alone, and N98S, Q332/333P, and E396/397K mutant NFL assembled in the presence of NFM and peripherin. P8R, P22S, N98S, L268/269P, E396/397K, and P440/441L mutant NFL co-assembled into neurofilaments with endogenous NFL, NFM, and α-internexin in cultured neurons, although the N98S and E396/397K mutants showed reduced filament incorporation, and the Q332/333P mutant showed limited incorporation. We conclude that all the mutants are capable of assembling into neurofilaments, but for some of the mutants this was dependent on the identity of the other neurofilament proteins available for co-assembly, and most likely also their relative expression level. Thus, caution should be exercised when drawing conclusions about the assembly capacity of CMT2E/1F mutants based on transient transfections in cultured cells.

Neurofilament heavy chain expression and neuroplasticity in rat auditory cortex after unilateral and bilateral deafness

Deafness induces many plastic changes in the auditory neural system. For instance, dendritic changes cause synaptic changes in neural cells. SMI-32, a monoclonal antibody reveals auditory areas and recognizes non-phosphorylated epitopes on medium- and high-molecular-weight subunits of neurofilament proteins in cortical pyramidal neuron dendrites. We investigated SMI-32-immunoreactive (-ir) protein levels in the auditory cortices of rats with induced unilateral and bilateral deafness. Adult male Sprague–Dawley rats were divided into unilateral deafness (UD), bilateral deafness (BD), and control groups. Deafness was induced by cochlear ablation. All rats were sacrificed, and the auditory cortices were harvested for real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analyses at 2, 4, 6, and 12 weeks after deafness was induced. Immunohistochemical staining was performed to evaluate the location of SMI-32-ir neurons. Neurofilament heavy chain (NEFH) mRNA expression and SMI-32-ir protein levels were increased in the BD group. In particular, SMI-32-ir protein levels increased significantly 6 and 12 weeks after deafness was induced. In contrast, no significant changes in protein level were detected in the right or left auditory cortices at any time point in the UD group. NEFH mRNA level decreased at 4 weeks after deafness was induced in the UD group, but recovered thereafter. Taken together, BD induced plastic changes in the auditory cortex, whereas UD did not affect the auditory neural system sufficiently to show plastic changes, as measured by neurofilament protein level.

Aberrant dendritic patterning in the visual cortex of MMP-3 deficient mice: effects on neurofilament proteins, CRMPs and experience-dependent plasticity.

Event Abstract Back to Event Aberrant dendritic patterning in the visual cortex of MMP-3 deficient mice: effects on neurofilament proteins, CRMPs and experience-dependent plasticity. Jeroen Aerts1, Julie Nys1, Lieve Moons2, Tjing-Tjing Hu1 and Lutgarde Arckens1* 1 KU Leuven, Biology, Belgium 2 KU Leuven, Biology, Belgium Matrix metalloproteinases (MMPs) constitute a multifunctional endopeptidase family that is essential for normal brain development and neuroplasticity by acting on extracellular matrix proteins, receptors, adhesion molecules, growth factors and cytoskeletal proteins. One member, MMP-3 has recently been implicated in synaptic plasticity, neuronal development and differentiation. However, the function of this enzyme in development and plasticity of the sensory neocortex remains understudied and therefore we characterized the visual cortex of MMP-3 deficient (MMP-3-/-) mice on an anatomical, molecular and functional level. Golgi-Cox staining revealed a significant reduction in apical dendritic length and dendritic spine length, as well as an increase in apical oblique number of layer V pyramidal neurons. In addition, MMP-3-/- mice displayed a significant upregulation of all known neurofilament protein subunits, indicative for aberrant cytoskeletal dynamics. Collapsin response mediator protein-5 (CRMP-5), a critical determinant for dendritic outgrowth was upregulated, thus further substantiating the aberrant dendritic patterning. To evaluate a possible role for CRMP-mediated changes in autophagy and mitophagy we observed an upregulation of LC3-II, a marker for autophagy, but no changes in Mfn-2 and Drp1, markers for mitochondrial fusion and fission respectively. Taken together, these data hint at a possible CRMP-mediated autophagy mechanism underlying the observed dendritic phenotype. To assess the effect of MMP-3 deficiency on cortical plasticity, adult mice were monocularly enucleated and analyzed for zif268-based recovery of neuronal activity in the contralateral visual cortex seven weeks post-enucleation. Neural activity was confined to the binocular zone and did not expand into the monocular regions, indicative for an aberrant open-eye potentiation. The permanent hypoactivity in the monocular cortex lateral and medial to V1 suggests a lack of cross-modal plasticity. Together, these observations corroborate that genetic removal of MMP-3 has profound effects on the structural and functional integrity of neurons, lasting into adulthood. Acknowledgements This work was supported by the Agency for Innovation through Science and Technology Flanders (IWT Vlaanderen). Keywords: Neurofilament, Golgi-Cox, MMP, pyramidal neuron, LC3-II, CRMPs, DRP1, Visual Cortex, neuroplasticity, Dendrites, Mfn-2 Conference: 11th National Congress of the Belgian Society for Neuroscience, Mons, Belgium, 22 May - 22 May, 2015. Presentation Type: Poster presentation Topic: Neuroscience Citation: Aerts J, Nys J, Moons L, Hu T and Arckens L (2015). Aberrant dendritic patterning in the visual cortex of MMP-3 deficient mice: effects on neurofilament proteins, CRMPs and experience-dependent plasticity.. Front. Neurosci. Conference Abstract: 11th National Congress of the Belgian Society for Neuroscience. doi: 10.3389/conf.fnins.2015.89.00002 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 05 May 2015; Published Online: 05 May 2015. * Correspondence: Dr. Lutgarde Arckens, KU Leuven, Biology, Leuven, 3000, Belgium, Lut.arckens@kuleuven.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jeroen Aerts Julie Nys Lieve Moons Tjing-Tjing Hu Lutgarde Arckens Google Jeroen Aerts Julie Nys Lieve Moons Tjing-Tjing Hu Lutgarde Arckens Google Scholar Jeroen Aerts Julie Nys Lieve Moons Tjing-Tjing Hu Lutgarde Arckens PubMed Jeroen Aerts Julie Nys Lieve Moons Tjing-Tjing Hu Lutgarde Arckens Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Neurofilaments as biomarkers in multiple sclerosis

Neurodegeneration is the correlate of disease progression in multiple sclerosis (MS) and thus biological biomarkers that sensitively reflect this process are much needed. Neurofilament protein subunits are potential cerebrospinal fluid (CSF) biomarkers for disease progression in MS. We argue that the neurofilament light subunit can reflect acute axonal damage mediated by inflammatory mechanisms and can imply prognostic value for conversion from clinically isolated syndrome (CIS) to definite MS. The neurofilament heavy subunit may rather reflect chronic irreversible damage and has prognostic value for disease progression or disability. The neurofilament intermediate subunit has not yet been studied. Recent studies showing higher neurofilament light or heavy subunit levels to be altered upon treatment regimes indicate their potential clinical value in monitoring treatment or side effects. Future studies should be aimed at the optimisation, standardisation and interlaboratory implementation of the assays and address the predictive value of these biomarkers.

Cytoarchitecture of the mouse neocortex revealed by the low-molecular-weight neurofilament protein subunit

The expression patterns of the medium- and high-molecular-weight subunits of the neurofilament protein triplet have been extensively studied in several neuroanatomical studies. In the present study, we report the use of the low-molecular-weight neurofilament protein subunit (NF-L) as a reliable marker within the neurofilament protein family to reveal the regional architecture of mammalian neocortex. We document clearly its usefulness in anatomical parcellation studies and report unique expression patterns of NF-L throughout the mouse neocortex. NF-L was most abundant in the somatosensory cortex, the lateral secondary visual area, the granular insular cortex, and the motor cortex. Low NF-L staining intensity was observed in the agranular insular cortex, the prelimbic and infralimbic cortex, the anterior cingulate cortex, the visual rostromedial areas, the temporal association cortex, the ectorhinal cortex, and the lateral entorhinal cortex. NF-L immunoreactivity was present in the perikarya, dendrites, and proximal segment of axons primarily of pyramidal neurons, and was mainly located in layers II and III, and to a lesser extent in layers V and VI. Interestingly, Black-Gold myelin staining confirmed a close correlation between NF-L immunoreactivity and myelination patterns. The characteristic and distinctive distribution and laminar expression profiles of NF-L make it an excellent tool to assess accurately topographical boundaries among neocortical areas as illustrated herein in the adult mouse brain.

Areas of cat auditory cortex as defined by neurofilament proteins expressing SMI-32

The monoclonal antibody SMI-32 was used to characterize and distinguish individual areas of cat auditory cortex. SMI-32 labels non-phosphorylated epitopes on the high- and medium-molecular weight subunits of neurofilament proteins in cortical pyramidal cells and dendritic trees with the most robust immunoreactivity in layers III and V. Auditory areas with unique patterns of immunoreactivity included: primary auditory cortex (AI), second auditory cortex (AII), dorsal zone (DZ), posterior auditory field (PAF), ventral posterior auditory field (VPAF), ventral auditory field (VAF), temporal cortex (T), insular cortex (IN), anterior auditory field (AAF), and the auditory field of the anterior ectosylvian sulcus (fAES). Unique patterns of labeling intensity, soma shape, soma size, layers of immunoreactivity, laminar distribution of dendritic arbors, and labeled cell density were identified. Features that were consistent in all areas included: layers I and IV neurons are immunonegative; nearly all immunoreactive cells are pyramidal; and immunoreactive neurons are always present in layer V. To quantify the results, the numbers of labeled cells and dendrites, as well as cell diameter, were collected and used as tools for identifying and differentiating areas. Quantification of the labeling patterns also established profiles for ten auditory areas/layers and their degree of immunoreactivity. Areal borders delineated by SMI-32 were highly correlated with tonotopically-defined areal boundaries. Overall, SMI-32 immunoreactivity can delineate ten areas of cat auditory cortex and demarcate topographic borders. The ability to distinguish auditory areas with SMI-32 is valuable for the identification of auditory cerebral areas in electrophysiological, anatomical, and/or behavioral investigations.

Tar DNA binding protein of 43 kDa (TDP-43), 14-3-3 proteins and copper/zinc superoxide dismutase (SOD1) interact to modulate NFL mRNA stability. Implications for altered RNA processing in amyotrophic lateral sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by progressive motor neuron degeneration in association with neurofilament (NF) aggregate formation. This process is accompanied by an alteration in the stoichiometry of NF subunit protein expression such that the steady state levels of the low molecular weight NF (NFL) mRNA levels are selectively suppressed. We have previously shown that each of TDP-43, 14-3-3 and mutant SOD1 can function as NFL mRNA 3′UTR binding proteins that directly affect the stability of NFL transcripts. In this study, we demonstrate that the interaction of TDP-43 with the NFL mRNA 3′ UTR involves ribonucleotide (UG) motifs present on stem loops of the 3′UTR as well as the RRM1 and RRM2 motifs of TDP-43. Ex vivo, TDP-43, 14-3-3 and SOD1 proteins interact to modulate NFL mRNA stability, although in vivo, only TDP-43 and either mutant or wild-type SOD1 co-localize in ALS motor neurons. TDP-43 was observed to co-localize to RNA transport granules (Staufen immunoreactive) in both control and ALS spinal motor neurons. In contrast, both stress granules (TIA-1 immunoreactive) and processing bodies (P-bodies; XRN-1 immunoreactive) were more prevalent in ALS motor neurons than in controls and demonstrated strong co-localization with TDP-43. Using RNA-IP-PCR, we further demonstrate that NFL mRNA is preferentially sequestered to both stress granules and P-bodies in ALS. These data suggest that NFL mRNA processing is fundamentally altered in ALS spinal motor neurons to favour compartmentalization within both stress granules and P-bodies, and that TDP-43 plays a fundamental role in this process.

Heterogeneous Distribution of Axonal Cytoskeleton Proteins in the Human Optic Nerve

Cytoskeleton proteins play a critical role in maintaining retinal ganglion cell structure, viability, and function. This study documents the distribution of cytoskeleton protein subunits in the various regions of the normal human optic nerve and identifies important relationships among mitochondria, myelin, and neurofilament proteins. Twenty-three optic nerves from human cadavers were used. Confocal microscopy was used to examine the distribution of neurofilament light, neurofilament medium, neurofilament heavy (phosphorylated and unphosphorylated), neurofilament heavy (phosphorylated only), actin, and microtubule associated protein (MAP)-1 along the sagittal plane of the optic nerve. Comparisons were made among superior, middle, and inferior regions and also among temporal, central, and nasal portions of the optic nerve. Colocalization of neurofilament light, mitochondrial cytochrome c oxidase (COX), and myelin was also performed. There are significant differences in the pattern and distribution of neurofilament protein subunits, actin, and MAP-1 along the sagittal plane of the optic nerve. Cytoskeleton proteins and COX mitochondria are found in highest concentrations in the prelaminar and lamina cribrosa regions. COX and neurofilament light occurs predominantly in unmyelinated nerve, with a significant decrease in concentration occurring on optic nerve myelination. The heterogeneous distribution of cytoskeleton proteins along the sagittal plane may be an important functional adaptation that reflects the nonuniform nature of the physiological and structural environment of the optic nerve. The heterogeneous distribution of cytoskeleton proteins may also partly account for the asymmetric pattern of optic nerve damage after intraocular pressure elevation.

Dynamic endogenous association of neurofilament mRNAs with K-homology domain ribonucleoproteins in developing cerebral cortex

The low, middle, and high molecular mass neurofilament subunit proteins (NF-L, NF-M, and NF-H) co-polymerize to form neurofilaments (NFs). During development, NF subunit expression is highly regulated, and in neurodegenerative disease, aberrant regulation of this expression can lead to the formation of harmful aggregates. NF expression in both development and disease is under significant post-transcriptional control, but the specific ribonucleoproteins (RNPs) involved are only poorly understood. Previously, mass spectrometry on affinity purified proteins from rat brain identified three K-homology (KH) domain RNPs – hnRNP K, hnRNP E1, hnRNP E2 – as being capable of binding NF-M RNA. In the current study, to determine whether these RNPs associate with NF mRNAs endogenously, we performed a co-immunoprecipitation assay on homogenates of postnatal and developing rat cerebral cortex. We found that all three NF mRNAs indeed associated endogenously with these RNPs and that the degree of this association changed during postnatal development, a period when NF expression is under significant post-transcriptional control. The degree of these associations changed independently of the abundance of either the RNPs or the NF messages, indicating that the RNA–protein interactions themselves are directly regulated. This study is consistent with a model whereby these RNPs and NF mRNAs are components of a dynamic post-transcriptional regulatory module that influences the cytoskeletal compositions of neurons.

Regional analysis of neurofilament protein immunoreactivity in the hamster's cortex

The laminar distribution of several distinct populations of neurofilament protein containing neurons has been used as a criterion for the delineation of cortical areas in hamsters. SMI-32 is a monoclonal antibody that recognizes a non-phosphorylated epitope on the medium- and high-molecular weight subunits of neurofilament proteins. As in carnivores and primates, SMI-32 immunoreactivity in the hamster neocortex was present in cell bodies, proximal dendrites and axons of some medium and large pyramidal neurons located in cortical layers III, V and VI. A small population of labeled multipolar cells was also found in layer IV. Neurofilament protein immunoreactive neurons were found throughout isocortical areas. Very few labeled cells were encountered in supplemental motor area, insular cortex, medial portion of associative visual cortex and in parietal association cortex. Our data indicate that SMI-32 immunoreactive cells can be efficiently used to trace boundaries between neocortical areas in the hamster's brain. The regional distribution SMI-32 immunoreactivity in the hamster cortex corresponds quite closely with cortical areas as defined by their cytoarchitecture and myeloarchitecture. The primary sensory cortical areas contain the most intense of SMI-32 immunoreactivity and are also those with the highest density of myelinated axons. Very low SMI-32 immunoreactivity was found in orbital, insular, perirhinal, cingulate and infralimbic cortices, which are also poor in myelinated axons. This supports the association between SMI-32 immunoreactivity and myelin contents.

2,5-Hexanedione induced reduction in protein content and mRNA expression of neurofilament in rat cerebral cortex

Exposure chronically to n-hexane produces central-peripheral axonopathy mediated by 2,5-hexanedione (HD). Studies have shown neurofilament (NF) subunit proteins are decreased substantially in cerebral cortices, optic axons, spinal cords, and sciatic nerves from HD-exposed rats. To deeply investigate the alterations in NF contents in HD neuropathy, the relative levels of NF-L, NF-M, and NF-H in rat cerebral cortex were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. HD was administrated to Wistar rats by intraperitoneal injection at dosage of 200 or 400 mg/kg. Rats were sacrificed after 6 weeks of treatment, and cerebral cortices were dissected, homogenized, and used for the determination of NF subunit proteins. The results, except for supernatant NF-L and NF-M that could not be assayed, showed HD intoxication resulted in significant decreases by 32–67% ( P < 0.01) in NF subunits in both of the pellet and supernatant fractions of cerebral cortex homogenate. As an initial investigation to determine how such changes in NF proteins might occur, the gene expression of NF-L, NF-M, and NF-H subunit mRNA was quantified using reverse transcription-polymerase chain reaction (RT-PCR). Statistical analysis revealed that HD exposure caused a significant reduction in the expression of NF-L and NF-H gene ( P < 0.05 or P < 0.01), while the levels of NF-M mRNA kept unaffected ( P > 0.05). These suggest that the observed reduction in NF gene expression might be related to diminished levels of subunit proteins, while the actual contribution might be uncertain. The functional significance of the reduced protein contents and the regulation of gene expression remain to be determined.

Neurofilament subunit protein abnormalities in the thalamus in schizophrenia

Neurofilament subunit protein abnormalities in the thalamus in schizophrenia

Neurofilament subunit protein abnormalities in the thalamus in schizophrenia

Postmortem and in vivo imaging studies have revealed structural and functional pathology in the thalamus in schizophrenia. We have reported a series neurochemical changes in the thalamus in schizophrenia, including decreased expression of NMDA receptor subunit transcripts and increased levels of NMDA receptor-associated intracellular proteins like postsynaptic density protein 95 (PSD95) and neurofilament-light (NF-L) chain that link the NMDA receptor to downstream signal transduction pathways. NF-L, along with the neurofilament subunit proteins NF-medium (NF-M) chain and NF-heavy (NF-H) chain, assemble to form neurofilaments, one of the most abundant cytoskeletal elements. While the neurofilament subunits play a crucial role in sustaining the neuronal cytoskeleton, two subunits, NF-L and NF-M, also participate in neurotransmission, interacting with the NMDA and the dopamine D1 receptors, respectively. In the present study, using in situ hybridization, we determined whether all of the neurofilament subunits (NF-L, NF-M, and NF-H) are abnormally expressed in the thalamus in schizophrenia, or whether the abnormality is specific to NF-L. We found that NF-L and NF-M transcripts are both increased in schizophrenia, but the expression of NF-H is unchanged. Both NF-L and NF-M play an important role in maintaining the neuronal cytoskeleton, so abnormal transcript expression may be associated with structural thalamic pathology. Alternatively, since these proteins are also known to interact with the NMDA and D1 dopamine receptors, abnormal transcript levels may be associated with abnormalities of neurotransmission in the thalamus in schizophrenia.

Identification of endogenous phosphorylation sites of bovine medium and low molecular weight neurofilament proteins by tandem mass spectrometry.

Neurofilament proteins (NFP) are intermediate filaments found in the neuronal cytoskeleton. They are highly phosphorylated, a condition that is believed to be responsible for the assembly and stability of the filaments. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) shows molecular masses for bovine NFP subunits of 63, 105, and 125 kDa for NFL, NFM, and NFH. Mass spectrometric de novo sequencing was used to determine the N-terminal sequence of bovine NFM (115 amino acids), which was previously unknown. Molecular mass information shows that there is one-half equivalent phosphate group on NFL and 24 on NFM. For the first time, it is shown that bovine NFL has three phosphorylation sites (Ser(55), Ser(66), and Ser(472)) and NFM has 22 (Ser(512), Ser(546), Ser(554), Ser(560), Thr(627), Ser(629), Ser(634), Ser(639), Thr(646), Ser(649), Ser(654), Ser(664), Ser(669), Thr(676), Ser(679), Ser(684), Ser(694), Ser(726), Ser(750), Ser(756), Ser(770), and Ser(846)) and two tentative sites (Ser(659)/Thr(661) and Thr(840)). Ser(66) was previously not known to be phosphorylated in NFL of other species, while two sites (Ser(55) and Ser(472)) are consistent with the phosphorylations observed in other mammalian NFLs. The three sites, Ser(55), Ser(66), Ser(472), are heterogeneously phosphorylated. Phosphorylation in bovine NFM occurs mainly in the Lys-Ser-Pro (KSP) region, but the Val-Ser-Pro and Ser-Glu-Lys motifs are also phosphorylated. Most of the phosphorylation sites are in accordance with those previously identified in other mammalian NFMs. In bovine NFM, 16 out of the 22 sites are always phosphorylated (Ser(512), Thr(627), Ser(629), Ser(634), Ser(639), Thr(646), Ser(649), Ser(654), Ser(664), Ser(669), Thr(676), Ser(679), Ser(684), Ser(694), Ser(726), and Ser(750)), all of which are contained in the KSP region, and six are sometimes phosphorylated (Ser(546), Ser(554), Ser(560), Ser(756), Ser(770), and Ser(846)). The NFPs have other modifications, including deamidation, oxidation, and N-terminal acetylation. Pyroglutamic acid formation also occurs.

Amino Acid and Protein Targets of 1,2-Diacetylbenzene, a Potent Aromatic γ-Diketone That Induces Proximal Neurofilamentous Axonopathy

The γ-diketone analogs 1,2-diacetylbenzene (1,2-DAB) and 2,5-hexanedione (2,5-HD), but not the δ-diketone 1,3-diacetylbenzene (1,3-DAB) or the β-diketone 2,4-hexanedione, induce neuropathological changes in the rodent central and peripheral nervous systems. The molecular targets of these neurotoxic aromatic and aliphatic γ-diketones, and of their nonneurotoxic structural analogs and ninhydrin, are examined by assessing their differential reactivity with neural and nonneural amino acids and proteins in vitro and in vivo. Whereas 1,2-DAB is chromogenic and forms polymers with amino acids (notably lysine) and proteins (especially lysine-rich proteins), 1,3-DAB lacks these properties. Ninhydrin forms a chromophore without evidence of protein polymerization. 1,2-DAB preferentially targets neurofilament over microtubule protein in vitro and in situ. Based on protein reactivity, 1,2-DAB is three orders of magnitude more reactive than 2,5-HD. Lysine-rich neurofilament protein subunits NF-H and NF-M are more susceptible than lysine-poor NF-L and β-tubulin to 1,2-DAB. These observations correlate with the development of proximal (1,2-DAB) and distal (2,5-HD) neurofilament-filled axonal swellings and segregated intact microtubules observed during systemic treatment with aromatic and aliphatic γ-diketones.

Phosphorylation of MEK1 by cdk5/p35 Down-regulates the Mitogen-activated Protein Kinase Pathway

Cyclin-dependent protein kinase 5 (cdk5), a member of the cdk family, is active mainly in postmitotic cells and plays important roles in neuronal development and migration, neurite outgrowth, and synaptic transmission. In this study we investigated the relationship between cdk5 activity and regulation of the mitogen-activated protein (MAP) kinase pathway. We report that cdk5 phosphorylates the MAP kinase kinase-1 (MEK1) in vivo as well as the Ras-activated MEK1 in vitro. The phosphorylation of MEK1 by cdk5 resulted in inhibition of MEK1 catalytic activity and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. In p35 (cdk5 activator) -/- mice, which lack appreciable cdk5 activity, we observed an increase in the phosphorylation of NF-M subunit of neurofilament proteins that correlated with an up-regulation of MEK1 and ERK1/2 activity. The activity of a constitutively active MEK1 with threonine 286 mutated to alanine (within a TPXK cdk5 phosphorylation motif in the proline-rich domain) was not affected by cdk5 phosphorylation, suggesting that Thr286 might be the cdk5/p35 phosphorylation-dependent regulatory site. These findings support the hypothesis that cdk5 and the MAP kinase pathway cross-talk in the regulation of neuronal functions. Moreover, these data and the recent studies of Harada et al. (Harada, T., Morooka, T., Ogawa, S., and Nishida, E. (2001) Nat. Cell Biol. 3, 453-459) have prompted us to propose a model for feedback down-regulation of the MAP kinase signal cascade by cdk5 inactivation of MEK1.

Neurofilament protein: a selective marker for the architectonic parcellation of the visual cortex in adult cat brain.

In this immunocytochemical study, we examined the expression profile of neurofilament protein in the cat visual system. We have used SMI-32, a monoclonal antibody that recognizes a nonphosphorylated epitope on the medium- and high-molecular-weight subunits of neurofilament proteins. This antibody labels primarily the cell body and dendrites of pyramidal neurons in cortical layers III, V, and VI. Neurofilament protein-immunoreactive neurons were prominent in 20 visual cortical areas (areas 17, 18, 19, 20a, 20b, 21a, 21b, and 7; posteromedial lateral, posterolateral lateral, anteromedial lateral, anterolateral lateral, dorsal lateral, ventral lateral, and posterior suprasylvian areas; anterior ectosylvian, the splenial, the cingulate, and insular visual areas; and the anterolateral gyrus area). In addition, we have also found strong immunopositive cells in the A laminae of the dorsal part of the lateral geniculate nucleus (dLGN) and in the medial interlaminar nucleus, but no immunoreactive cells were present in the parvocellular C (1-3) laminae of the dLGN, in the ventral part of the LGN and in the perigeniculate nucleus. This SMI-32 antibody against neurofilament protein revealed a characteristic pattern of immunostaining in each visual area. The size, shape, intensity, and density of neurofilament protein-immunoreactive neurons and their dendritic arborization differed substantially across all visual areas. Moreover, it was also obvious that several visual areas showed differences in laminar distribution and that such profiles may be used to delineate various cortical areas. Therefore, the expression of neurofilament protein can be used as a specific marker to define areal patterns and topographic boundaries in the cat visual system.