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PubMed2002

alpha-Synuclein protects against oxidative stress via inactivation of the c-Jun N-terminal kinase stress-signaling pathway in neuronal cells.

The expression of alpha-synuclein, a synaptic molecule implicated in the pathogenesis of neurodegenerative disorders such as Parkinson's disease and Lewy body disease is increased upon injury to the nervous system, indicating that it might play a role in regeneration and plasticity; however, the mechanisms are unclear. Because c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, plays an important role in stress response, the main objective of the present study was to better understand the involvement of this pathway in the signaling responses associated with resistance to injury in cells expressing alpha-synuclein. For this purpose, the JNK-signaling pathway was investigated in alpha-synuclein-transfected neuronal cell line glucose transporter (GT) 1-7 under oxidative stress conditions. Although hydrogen peroxide challenge resulted in JNK activation and cell death in cells transfected with vector control or beta-synuclein, alpha-synuclein-transfected cells were resistant to hydrogen peroxide, and JNK was not activated. The inactivation of JNK in the alpha-synuclein-transfected cells was associated with increased expression and activity of JNK-interacting protein (JIP)-1b/islet-brain (IB)1, the scaffold protein for the JNK pathway. Similarly, cells transfected with JIP-1b/IB1 were resistant to hydrogen peroxide associated with inactivation of the JNK pathway. In these cells, expression of endogenous alpha-synuclein was significantly increased at the protein level. Furthermore, alpha-synuclein was co-localized with JIP-1b/IB1 in the growth cones. Taken together, these results suggest that increased alpha-synuclein expression might protect cells from oxidative stress by inactivation of JNK via increased expression of JIP-1b/IB1. Furthermore, interactions between alpha-synuclein and JIP-1b/IB1 may play a mutual role in the neuronal response to injury and neurodegeneration.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Oxidative stress induces amyloid-like aggregate formation of NACP/alpha-synuclein in vitro.

The precursor of non-amyloid beta protein component of Alzheimer's disease amyloid (NACP/alpha-synuclein), found in Lewy bodies of Parkinson's disease (PD), is a presynaptic protein genetically linked to some familial types PD. Mechanisms of abnormal NACP/alpha-synuclein aggregation in neurodegenerative diseases are unclear. Since oxidative stress might play a role in PD pathogenesis, we investigated the role of iron and peroxide in NACP/alpha-synuclein aggregation. Immunoblot analysis showed that human NACP/alpha-synuclein (but not beta-synuclein) aggregated in the presence of ferric ion and was inhibited by the iron chelator deferoxamine. Ferrous ion was not effective by itself, but it potentially aggregated NACP/alpha-synuclein in the presence of hydrogen peroxide. NACP/ alpha-synuclein aggregates displayed strong thioflavine-S and congo-red reactivity, reminiscent of amyloid. This study suggests that NACP/alpha-synuclein aggregation might be closely related to oxidative reactions which may play a critical role in neurodegeneration in disorders with Lewy bodies.

Alzheimer Disease↗
PubMed2002

Evidence that alpha-synuclein functions as a negative regulator of Ca(++)-dependent alpha-granule release from human platelets.

alpha-Synuclein has been implicated in the pathogenesis of Parkinson disease (PD) and related neurodegenerative disorders. More recently, it has been suggested to be an important regulatory component of vesicle transport in neuronal cells. alpha-Synuclein is also highly expressed in platelets and is loosely associated with the membrane of the secretory alpha-granules. However, the functional significance of these observations is unknown. In this study, the possible function of alpha-synuclein in vesicle transport, with particular regard to alpha-granule release from the platelets, was investigated. The results showed that ionomycin- or thrombin-induced alpha-granule secretion was inhibited by exogenous alpha-synuclein addition in a dose-dependent manner. However, [(3)H]5-HT release from the dense granules and hexosaminidase release from the lysosomal granules were not affected. Two point mutants (A30P and A53T) found in some familial types of PD, in addition to beta-synuclein and alpha-synuclein112, effectively inhibited PF4 release from the alpha-granules. However, the deletion mutants, which completely lacked either the N-terminal region or the C-terminal tail, did not affect alpha-granule release. Interestingly, exogenously added alpha-synuclein appeared to enter the platelets but did not change the Ca(++) level in the platelets at the resting state and the increase in the Ca(++) level on stimulation. Electron microscopy also supported that alpha-synuclein inhibits alpha-granule release. These results suggest that alpha-synuclein may function as a specific negative regulator of alpha-granule release in platelets.

Blood Platelets↗
PubMed2001

Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice.

BACKGROUND: The presynaptic protein alpha-synuclein is involved in a range of neurodegenerative diseases. Here we analyze potential compensatory mechanisms in alpha-synuclein null mutant mice. Furthermore, the findings reveal problems that may be associated with inbred mouse strains. RESULTS: Expression profiling by cDNA array technology in a transgenic mouse model revealed striking differences only in the expression level of alpha-synuclein. This was caused by a chromosomal deletion of the alpha-synuclein locus in the C57BL/6J inbred strain used for backcrossing. However, the deletion is only present in a subpopulation of C57BL/6J mice, namely animals from Harlan. No other genes are known to be affected by the deletion, which is estimated to be smaller than 2 cM. We propose to name this strain C57BL/6S. C57BL/6S animals appear phenotypically normal. They show no upregulation of beta-synuclein or gamma-synuclein, excluding a compensatory mechanism. Also, the expression of synphilin-1 was unaffected. CONCLUSIONS: The C57BL/6S strain should help in the understanding of the physiological function of alpha-synuclein and its involvement in synucleinopathies. Also, the findings exemplify unexpected complications that may arise during the study of transgenic models or inbred strains, in particular when combined with genome wide screening techniques.

Animals↗
PubMed2001

Parkinson's disease and other alpha-synucleinopathies.

Parkinson's disease is the most common movement disorder and the second most common neurodegenerative disease. Neuropathologically, it is characterized by the degeneration of nerve cells that develop filamentous inclusions in the form of Lewy bodies and Lewy neurites. Recent work has shown that rare, familial forms of Parkinson's disease are caused by missense mutations in the alpha-synuclein gene and that the filamentous lesions of Parkinson's disease are made of alpha-synuclein. The same is true of the Lewy body pathology that is associated with other neurodegenerative diseases, such as dementia with Lewy bodies. The filamentous inclusions of multiple system atrophy have also been found to be made of alpha-synuclein, thus providing an unexpected molecular link with Lewy body diseases. Recombinant alpha-synuclein assembles into filaments with similar morphologies to those found in the human diseases and with a cross-beta diffraction pattern characteristic of amyloid. The related proteins beta-synuclein and gamma-synuclein are poor at assembling into filaments. They are not present in the pathological filamentous lesions and have not been found to be linked to genetic disease. The new work has established the alpha-synucleinopathies as a major class of neurodegenerative disease.

Animals↗
PubMed1999

[Parkinson's disease, dementia with Lewy bodies, multiple system atrophy and alpha-synuclein].

Lewy bodies (LBs) are hallmark lesions of degenerating neurons in the brains of patients with Parkinson's disease (PD) and dementia with Lewy bodies (DLB). DLB is the second most common neurodegenerative dementia after Alzheimer's disease, which is characterized clinically by fluctuating cognitive impairments, visual hallucinations and parkinsonism, and pathologically by the appearance of cortical LBs. To characterize the components of LBs, we have developed a purification procedure for LBs from cortices of patients with DLB using sucrose density separation followed by fluorescence-activated particle sorting. We then raised monoclonal antibodies (mAbs) to purified LBs, and obtained a mAb (LB509) that intensely immunolabeled LBs and specifically reacted with a approximately 18kDa brain protein, which was identified as alpha-synuclein. LB509 as well as other antibodies to alpha-synuclein, but not to beta-synuclein, immunostained brainstem and cortical LBs in sporadic PD and DLB brains. Recently, a point mutation in alpha-synuclein gene was identified in some autosomal-deminantly inherited familial PD pedigrees. Moreover, glial cytoplasmic inclusions in the brains of patients with multiple system atrophy (MSA) were shown to be alpha-synuclein positive. Taken together, our data strongly implicate alpha-synuclein in the formation of LBs and the selective neuronal degeneration in PD, DLB and MSA.

Humans↗
PubMed2001

Induction of alpha-synuclein aggregation by intracellular nitrative insult.

Brain lesions containing filamentous and aggregated alpha-synuclein are hallmarks of neurodegenerative synucleinopathies. Oxidative stress has been implicated in the formation of these lesions. Using HEK 293 cells stably transfected with wild-type and mutant alpha-synuclein, we demonstrated that intracellular generation of nitrating agents results in the formation of alpha-synuclein aggregates. Cells were exposed simultaneously to nitric oxide- and superoxide-generating compounds, and the intracellular formation of peroxynitrite was demonstrated by monitoring the oxidation of dihydrorhodamine 123 and the nitration of alpha-synuclein. Light microscopy using antibodies against alpha-synuclein and electron microscopy revealed the presence of perinuclear aggregates under conditions in which peroxynitrite was generated but not when cells were exposed to nitric oxide- or superoxide-generating compounds separately. alpha-Synuclein aggregates were observed in 20-30% of cells expressing wild-type or A53T mutant alpha-synuclein and in 5% of cells expressing A30P mutant alpha-synuclein. No evidence of synuclein aggregation was observed in untransfected cells or cells expressing beta-synuclein. In contrast, selective inhibition of the proteasome resulted in the formation of aggregates detected with antibodies to ubiquitin in the majority of the untransfected cells and cells expressing alpha-synuclein. However, alpha-synuclein did not colocalize with these aggregates, indicating that inhibition of the proteasome does not promote alpha-synuclein aggregation. In addition, proteasome inhibition did not alter the steady-state levels of alpha-synuclein, but addition of the lysosomotropic agent ammonium chloride significantly increased the amount of alpha-synuclein, indicating that lysosomes are involved in degradation of alpha-synuclein. Our data indicate that nitrative and oxidative insult may initiate pathogenesis of alpha-synuclein aggregates.

Ammonium Chloride↗
PubMed2002

Synucleins in glaucoma: implication of gamma-synuclein in glaucomatous alterations in the optic nerve.

Synucleins are small proteins associated with neurodegenerative diseases and some forms of cancer. They are studied predominantly in the brain; information about their presence and functions in ocular tissues is scarce. Here we describe the localization of three members of the synuclein family in the optic nerve of donors with different types of glaucoma compared with control samples from donors without ocular diseases. We did not find significant differences in the localization of alpha- and beta-synucleins in the optic nerve or retina of glaucoma patients compared with controls, whereas considerable redistribution of gamma-synuclein occurred in the glaucomatous optic nerve compared with control eye without glaucoma. In the optic nerve from control and glaucomatous individuals, nerve bundles are immunopositive for gamma-synuclein; however, a strong gamma-synuclein-immunopositive staining in a subset of glial cells was observed in the lamina and postlamina cribrosa regions of the optic nerve only in glaucoma patients. In the optic nerve of rats with episcleral vein cauterization used as an animal model of glaucoma, the quantity of both gamma-synuclein mRNA and protein was decreased compared with the optic nerves of control animals. Incubation of rat astrocyte culture at elevated hydrostatic pressure reduced the amount of gamma-synuclein but did not affect the quantities of actin and glial fibrillary acidic protein. These data suggest that significant changes in the pattern of expression and/or localization occur in the glaucomatous optic nerve for gamma-synuclein but not for alpha- and beta-members of the synuclein family.

Aged↗
PubMed2001

Synucleins in ocular tissues.

Synucleins are small proteins associated with neurodegenerative diseases and some forms of cancer. Most studies of this group of proteins have been directed to the elucidation of their role in the brain and their connection to the formation of depositions in brain tissues. Here we describe the localization of different types of synucleins in ocular tissues. By Western blot analysis, all members of the synuclein family are found in the retina and optic nerve, where their relative ratio varies. The data on immunohistochemical staining show that different members of the synuclein family have different localizations in ocular tissues. Alpha-synucleins and beta-synucleins are present predominantly in the inner plexiform layer, whereas gamma-synuclein is in the nerve fiber layer. In transgenic mice overexpressing alpha-synuclein, a different pattern of localization depending on the promoter used for the expression was observed. In Alzheimer's disease patients, immunohistochemical staining for gamma-synuclein revealed the loss of immunoreactivity in the nerve fiber layer and the nerve fiber layer and the appearance of immunopositive cells in or near the outer nuclear layer. We conclude that, in mature eyes, synucleins are present predominantly in the retina and optic nerve, and the immunoreactivity of gamma-synuclein changes specifically in the retina of Alzheimer's disease patients. In transgenic mice overexpressing alpha-synuclein, immunopositive deposits in the optic nerve and accumulation of immunoreactivity in specific retinal cells were found.

Aged↗
PubMed1999

The expression of alpha-, beta-, and gamma-synucleins in olfactory mucosa from patients with and without neurodegenerative diseases.

A family of homologous proteins known as alpha-, beta-, and gamma-synuclein are abundantly expressed in brain, especially in the presynaptic terminal of neurons. Although the precise function of these proteins remains unknown, alpha-synuclein has been implicated in synaptic plasticity associated with avian song learning as well as in the pathogenesis of Parkinson's disease (PD), dementia with LBs (DLB), some forms of Alzheimer's disease (AD), and multiple system atrophy (MSA). Since olfactory dysfunction is a common feature of these disorders and the olfactory receptor neurons (ORNs) of the olfactory epithelium (OE) regenerate throughout the lifespan, we used antibodies specific for alpha-, beta-, and gamma-synucleins to examine the olfactory mucosa of patients with PD, DLB, AD, MSA, and controls without a neurological disorder. Although antibodies to alpha- and beta-synucleins detected abnormal dystrophic neurites in the OE of patients with neurodegenerative disorders, similar pathology was also seen in the OE of controls. More significantly, we show here for the first time that alpha-, beta-, and gamma-synucleins are differentially expressed in cells of the OE and respiratory epithelium and that alpha-synuclein is the most abundant synuclein in the olfactory mucosa, where it is prominently expressed in ORNs. Moreover, alpha- and gamma-synucleins also were prominent in the OE basal cells, which include the progenitor cells of the ORNs in the OE. Thus, our data on synuclein expression within the OE may signify that synuclein plays a role in the regeneration and plasticity of ORNs in the adult human OE.

Adult↗
PubMedDate not supplied

Quantitative neurohistological features of frontotemporal degeneration.

Frontotemporal degeneration (FTD) is a neurodegenerative condition that has been principally associated with frontal lobe dementia. In this study, we compared neuropathological abnormalities in frontal, hippocampal, and calcarine cortices from patients assigned a diagnosis of FTD, normal elderly and Alzheimer's disease (AD). Densities of Nissl-stained neurons and lesions which were immunolabeled for tau, beta-amyloid (Abeta), alpha- and beta-synuclein, ubiquitin, glial fibrillary acidic protein (GFAP) and CD68 antigen were determined using computer-assisted, non-biased quantitative microscopy. We found that FTD frontal and hippocampal regions exhibited marked neuron loss, abundant ubiquitin-immunoreactive (ir) dystrophic neurites, GFAP-ir astrocytes, and CD68-ir microglia, while calcarine cortex was spared. No alpha- or beta-synuclein-ir lesions were observed, and neither the density of tau-ir neurofibrillary tangles nor that of Abeta-ir plaques in FTD exceeded normal controls. In addition, there were no neuropathological differences between FTD subjects who presented clinically with a frontal lobe dementia versus an AD-like dementia. These findings indicate that FTD is a category of neurodegnerative dementias with varying clinical presentations that is characterized by the progressive degeneration of select populations of cortical neurons. The molecular neurodegenerative mechanisms that lead to FTD remain to be elucidated.

Aged↗
PubMed2001

Cytosolic O-glycosylation is abundant in nerve terminals.

Phosphorylation plays a key role in regulating growth cone migration and protein trafficking in nerve terminals. Here we show that nerve terminal proteins contain another abundant post-translational modification: beta-N-acetylglucosamine linked to hydroxyls of serines or threonines (O-GlcNAc(1)). O-GlcNAc modifications are essential for embryogenesis and mounting evidence suggests that O-GlcNAc is a regulatory modification that affects many phosphorylated proteins. We show that the activity and expression of O-GlcNAc transferase (OGT) and N-acetyl-beta-D-glucosaminidase (O-GlcNAcase), the two enzymes regulating O-GlcNAc modifications, are present in nerve terminal structures (synaptosomes) and are particularily abundant in the cytosol of synaptosomes. Numerous synaptosome proteins are highly modified with O-GlcNAc. Although most of these proteins are present in low abundance, we identified by proteomic analysis three neuron-specific O-GlcNAc modified proteins: collapsin response mediator protein-2 (CRMP-2), ubiquitin carboxyl hydrolase-L1 (UCH-L1) and beta-synuclein. CRMP-2, which is involved in growth cone collapse, is a major O-GlcNAc modified protein in synaptosomes. All three proteins are implicated in regulatory cascades that mediate intracellular signaling or neurodegenerative diseases. We propose that O-GlcNAc modifications in the nerve terminal help regulate the functions of these and other synaptosome proteins, and that O-GlcNAc may play a role in neurodegenerative disease.

Acetylglucosamine↗
PubMed2001

Focal inhibitory interneuron loss and principal cell hyperexcitability in the rat hippocampus after microinjection of a neurotoxic conjugate of saporin and a peptidase-resistant analog of Substance P.

Episodes of prolonged seizures or head trauma produce chronic hippocampal network hyperexcitability hypothesized to result primarily from inhibitory interneuron loss or dysfunction. The possibly causal role of inhibitory neuron failure in the development of epileptiform pathophysiology remains unclear because global neurologic injuries produce such a multitude of effects. The recent finding that Substance P receptors (SPRs) are expressed exclusively in the rat hippocampus by inhibitory interneurons provided the rationale for attempting to ablate interneurons selectively by using neurotoxic conjugates of SPR ligands and the ribosome inactivating protein saporin that specifically target Substance P receptor-expressing cells. Whereas intrahippocampal microinjection of a conjugate of native SP and saporin produced significant nonspecific damage at concentrations needed to produce even limited selective loss of SPR-positive cells, a conjugate of saporin and the more potent and peptidase-resistant SP analog [Sar(9), Met(O(2))(11)] Substance P (SSP-saporin) caused negligible nonspecific damage at the injection site, and a virtually complete loss of SPR-like immunoreactivity (LI) up to 1 mm from the injection site. Within the SPR depletion zone, immunoreactivities for most GABA-, parvalbumin-, somatostatin-, and cholecystokinin-immunoreactive cells and fibers were eliminated. The few interneurons detectable within the affected zone were devoid of SPR-LI. The apparent loss of interneurons was selective in that calbindin- and glutamate receptor subunit 2 (GluR2) -positive principal cells survived within the affected zone, as did myelinated fibers and the extrinsic calretinin- and tyrosine hydroxylase--immunoreactive terminals of subcortical afferents. An apparent lack of reactive synaptic reorganization in response to interneuron loss was indicated by zinc transporter-3 (ZnT3)-- and beta-synuclein--LI, as well as by Timm staining, all of which revealed relatively normal patterns of excitatory terminal distribution. Control injections produced minor damage at the injection site, but no apparent specific loss of SPR-LI. One to 12 weeks after injection of SSP-saporin, extracellular electrophysiological field responses recorded in the CA1 pyramidal and dentate granule cell layers in response to afferent stimulation were blindly evaluated simultaneously in two sites 1-2 mm apart along the longitudinal hippocampal axis. SSP-saporin-treated rats exhibited relatively normal responses in some sites, whereas disinhibition and hyperexcitability indistinguishable from the pathophysiology produced by experimental status epilepticus were simultaneously recorded at adjacent sites. Anatomic analysis of the recording sites in each animal revealed that epileptiform pathophysiology was consistently observed only within areas of SPR ablation, whereas relatively normal evoked responses were recorded from immediately adjacent and relatively unaffected regions. These data establish the efficacy of [Sar(9), Met(O(2))(11)] Substance P-saporin for producing a selective and spatially extensive ablation of hippocampal inhibitory interneurons in vivo and a highly focal disinhibition that was restricted to the site of interneuron loss. These results also demonstrate that the "epileptic" pathophysiology produced by experimental status epilepticus or head trauma can be replicated by focal interneuron loss per se, without involving principal cell loss and other interpretive confounds inherent in the use of global neurologic injury models.

Animals↗
PubMed2002

Pick's disease: alpha- and beta-synuclein-immunoreactive Pick bodies in the dentate gyrus.

Recent studies have shown that neurofibrillary tangles frequently coexist with alpha-synuclein (alpha-S)-positive fibrillary inclusions in the limbic system in Alzheimer's disease. To elucidate whether alpha-, beta- and gamma-S immunoreactivity is present in Pick bodies (PBs), we examined immunohistochemically and immunoelectron microscopically the brains from three patients with Pick's disease. Numerous PBs were distributed widely, and were occasionally immunoreactive for alpha-S and beta-S, but not for gamma-S in all three cases. However, these immunoreactive PBs were almost all restricted to the dentate gyrus. Despite the co-localization of phosphorylated tau and alpha-S or beta-S (as evidenced by double-labeling immunohistochemistry), immunoelectron microscopy revealed that alpha-S and beta-S immunoreactivity occurs in granular and vesicular structures, but not in filamentous structures. These findings suggest that alpha-S and beta-S are up-regulated in the neuronal perikarya but they are not incorporated into the constituent filaments of PBs, and that the preferential distribution of alpha-S- and beta-S-positive PBs in the dentate gyrus may represent the cellular response to PB formation in this particular system.

Aged↗
PubMed1993

Cell and tissue distribution and developmental change of neuron specific 14 kDa protein (phosphoneuroprotein 14).

In the present paper, the distribution of a neuron-specific phosphoneuroprotein 14 (PNP 14) in cell and tissue was investigated in detail by the immunoblot method using affinity-purified antibody against this protein. The immunoblot of the supernatant fractions of various tissue homogenates of rat clearly demonstrated that PNP 14 was enormously rich in the brain. The content in rat brain was as much as 0.1% of the homogenate. The immunocytochemical study showed that the protein was localized at nerve endings in the cerebellum. Existence of the protein was also confirmed in cultured neuronal cells from postnatal rat midbrain, but not in glial cells. Examination of subcellular localization of PNP 14 indicates that the protein was present in synaptic plasma membranes and synaptic supernatant fractions, but not in synaptic vesicles. During the development of rat brain, PNP 14 came into existence after birth and it's amount linearly increased to a maximum at 21-28 days after birth. The content of the protein then remained at the same level for more than 10 months. We concluded that this protein is neuron specific and supposed that it may be involved in neuronal formation and function.

Amino Acid Sequence↗
PubMed1994

Localization of phosphoneuroprotein 14 (PNP 14) and its mRNA expression in rat brain determined by immunocytochemistry and in situ hybridization.

Distribution and ultrastructural localization of a novel phosphoneuroprotein with a molecular mass of 14 kDa (PNP 14), and expression of its mRNA were studied in the adult rat central nervous system (CNS) by immunocytochemistry and in situ hybridization histochemistry. PNP 14 immunoreactivity was abundant in the molecular layer, present moderately in the granular layer, and rare in the Purkinje cell layer of the cerebellar cortex. No PNP 14 immunoreactivity was detected in the cerebellar medulla. In the forebrain, immunoreactivity was found in the hippocampus, striatum, and throughout the cerebral cortex, especially in layer V. Electron microscopic immunocytochemical observation in the cerebellar cortex revealed many PNP 14-immunoreactive axon terminals making synaptic contact with dendritic processes in both granular and molecular layers. PNP 14 immunoreactivity was present mainly in the cytoplasmic matrix in the presynaptic axon terminals. PNP 14 mRNA was localized in the granular layer of the cerebellar cortex, in the hippocampus, and in the cerebral cortex, suggesting that PNP 14 is synthesized in neurons of the granular layer and then transported to the molecular layer by axonal transport. These morphological findings suggest that PNP 14 is likely to modulate the function of selected CNS synapses.

Animals↗
PubMed1996

Distribution of phosphoneuroprotein 14 (PNP 14) in vertebrates: its levels as determined by enzyme immunoassay.

We have established an enzyme immunoassay for phosphoneuroprotein 14 (PNP 14) which is mainly localized in the cytoplasmic matrix in presynaptic axon terminals and which is phosphorylated in vivo, as well as in vitro. Fab' prepared from rabbit IgG antibodies against bovine PNP 14 was conjugated with maleimide-horseradish peroxidase. The enzyme-conjugated Fab' was used as a second antibody in a sandwich enzyme immunoassay. This assay was able accurately to quantify 0.5-100 ng of rat PNP 14, as well as bovine PNP 14, and it was used for the determination of concentrations of PNP 14 in various rat tissues, neuroblastoma cells, and brains of other vertebrates. The concentrations of PNP 14 in the rat cerebrum, cerebellum, and testis were 1.1, 1.0, and 0.28 micrograms/mg of protein, respectively, and those in other tissues examined were less than 0.1 microgram/mg of protein. PNP 14 was also found in cultured cells, such as rat pheochromocytoma PC12 cells, NG108-15 cells, which are a hybrid between a mouse neuroblastoma and a rat glioma, mouse neuroblastoma Neuro-2a cells, and human neuroblastoma IMR32 cells. Furthermore, PNP 14-specific immunoreactivity was evaluated in the brains of various vertebrates, such as fish, frog, snake and chicken by immunoblot and enzyme immunoassay. The results revealed the immunoreactivity in the brains of all vertebrates examined and the levels were determined to be 0.6-2.1 micrograms bovine PNP 14 equivalents per mg of protein, suggesting that PNP 14 might be an essential component of the central nervous systems of vertebrates.

Animals↗
PubMed1993

A new brain-specific 14-kDa protein is a phosphoprotein. Its complete amino acid sequence and evidence for phosphorylation.

We previously reported a new brain-specific protein with a molecular mass of 14 kDa, specifically present in synapses around neurons but not in glial cells [Nakajo, S., Omata, K., Aiuchi, T., Shibayama, T., Okahashi, I., Ochiai, H., Nakai, Y., Nakaya, K. & Nakamura, Y. (1990) J. Neurochem. 55, 2031-2038]. In the present study, we determined the primary structure of this protein, found that it is phosphorylated in vitro and in vivo, and designated it phosphoneuroprotein 14 (PNP 14). The protein is a single polypeptide with 134 amino acid residues (molecular mass = 14122 Da), and it contains a hydrophobic region at the center of the molecule. The carboxy-terminal region has all seven proline residues, and is rich in glutamic acid, which contribute to the acidic property of the protein. The amino-terminal region possesses four unique repetitive motifs, Glu(Ser)-Lys-Thr-Lys-Glu(Gln)-Gly(Gln)-Val(Ala). When a cytosolic fraction prepared from rat cerebral cortex was incubated with [gamma-32P]ATP, 32P was incorporated into PNP 14. Phosphorylated PNP 14 was immunoprecipitated from rat brain synaptosomes labeled metabolically with [32P]orthophosphate. Injection of [32P]orthophosphate into the third ventricle of rat brain resulted in incorporation of radioactive phosphate into PNP 14. We have also found that Ca2+, calmodulin-dependent protein kinase II phosphorylates serine residue(s) of PNP 14 in vitro. The results suggest that PNP 14 may be important to neuronal cells, but not to glial cells, and that its physiological functions may be controlled by the phosphorylation reaction.

Amino Acid Sequence↗