Cobalamin and normal prions: A new horizon for cobalamin neurotrophism

It is known that cobalamin (Cbl) deficiency damages myelin by increasing tumor necrosis factor (TNF)-α and decreasing epidermal growth factor (EGF) levels in rat central nervous system (CNS), and affects the peripheral nervous system (PNS) morphologically and functionally. It is also known that some polyneuropathies not due to Cbl deficiency are connected with increased TNF-α levels, and that various cytokines (including TNF-α) and growth factors regulate the in vitro synthesis of normal prions (PrP^Cs). Given that there is extensive evidence that PrP^Cs play a key role in the maintenance of CNS and PNS myelin, we investigated whether the PrP^C octapeptide repeat (OR) region is involved in the pathogenesis of rat Cbl-deficient (Cbl-D) polyneuropathy. After intracerebroventricularly administering antibodies (Abs) against the OR region (OR-Abs) to Cbl-D rats to prevent myelin damage and maximum nerve conduction velocity (MNCV) abnormalities, and PrP^Cs to otherwise normal rats to reproduce PNS Cbl-D-like lesions, we measured PrP^C levels and MNCV of the sciatic and tibial nerves. PrP^C and TNF-α levels were increased in sciatic and tibial nerves of Cbl-D and saline-treated rats, and the OR-Abs normalized the myelin ultrastructure, TNF-α levels, and MNCV values of the sciatic and tibial nerves of Cbl-D rats. The same peripheral nerves of the otherwise normal PrP^C-treated rats showed typical Cbl-D myelin lesions, significantly increased TNF-α levels, and significantly decreased MNCV values. These findings demonstrate that Cbl deficiency induces excess PrP^Cs and thereby excess OR regions, which seem to be responsible for the PNS myelin damage, as has recently been found in the case of CNS myelin damage [66]. Furthermore, excess TNF-α is also involved in the pathogenesis of Cbl-D polyneuropathy. In conclusion, we have extended the list of prion diseases by adding one caused by excess PrP^Cs and the polyneuropathies related to excess TNF-α.     

Epidermal growth factor as a local mediator of the neurotrophic action of vitamin B(12) (cobalamin) in the rat central nervous system.

We have recently demonstrated that the myelinolytic lesions in the spinal cord (SC) of rats made deficient in vitamin B(12) (cobalamin) (Cbl) through total gastrectomy (TG) are tumor necrosis factor-alpha (TNF-alpha)-mediated. We investigate whether or not permanent Cbl deficiency, induced in the rat either through TG or by chronic feeding of a Cbl-deficient diet, might modify the levels of three physiological neurotrophic factors-epidermal growth factor (EGF), vasoactive intestinal peptide (VIP), and somatostatin (SS)-in the cerebrospinal fluid (CSF) of these rats. We also investigated the ability of the central nervous system (CNS) in these Cbl-deficient rats to synthesize EGF mRNA and of the SC to take up labeled Cbl in vivo. Cbl-deficient rats, however the vitamin deficiency is induced, show a selective decrease in EGF CSF levels and an absence of EGF mRNA in neurons and glia in various CNS areas. In contrast, radiolabeled Cbl is almost exclusively taken up by the SC white matter, but to a much higher degree in totally gastrectomized (TGX) rats. Chronic administration of Cbl to TGX rats restores to normal both the EGF CSF level and EGF mRNA expression in the various CNS areas examined. This in vivo study presents the first evidence that the neurotrophic action of Cbl in the CNS of TGX rats is mediated by stimulation of the EGF synthesis in the CNS itself. It thus appears that Cbl inversely regulates the expression of EGF and TNF-alpha genes in the CNS of TGX rats.     

Cobalamin-mediated regulation of transcobalamin receptor levels in rat organs

Total gastrectomy (TG) causes cobalamin (Cbl) deficiency followed by increases in tumor necrosis factor (TNF)-alpha levels in the spinal cord (SC) of the rat. In order to understand how Cbl deficiency may influence cell Cbl transport, we have measured by immunoblotting protein levels of the receptor for the Cbl-transcobalamin (TC) complex (TC-R) in both animal and cell models. TC-R protein levels were elevated in the total membranes of duodenal mucosa, kidneys, liver, and SC of rats made Cbl-deficient (Cbl-D) by means of TG or feeding with a Cbl-D diet. Postoperative Cbl-replacement treatment normalized the TC-R protein levels in each of the tested organs, regardless of whether this treatment was given during the first two post-TG or during the third and fourth post-TG mo. In Caco-2 cells, progressively increasing TNF-alpha concentrations supplemented to culture medium induced an up-regulation of TC-R protein levels. We provide the first evidence of the regulation of a Cbl-specific receptor by the vitamin itself in some rat organs.     

Reduction of PrPC in human cerebrospinal fluid after spinal cord injury

It has been estimated that cerebrospinal fluid (CS F) contains approximately 80 proteins that significantly increase or decrease in response to various clinical conditions. Here we have evaluated the CS F protein PrP^C (cellular prion protein) for possible increases or decreases following spinal cord injury. The physiological function of PrP^C is not yet completely understood; however, recent findings suggest that PrP^C may have neuroprotective properties. Our results show that CS F PrP^C is decreased in spinal cord injured patients 12 h following injury and is absent at 7 days. Given that normal PrP^C has been proposed to be neuroprotective, we speculate that the decrease in CS F PrP^C levels may influence neuronal cell survival following spinal cord injury.Key words: CSF, PrP^C, Hsp25, crystallin domain, spinal cord injury     

New insights into the pathophysiology of cobalamin deficiency

Cobalamin-deficient (Cbl-D) central neuropathy in the rat is associated with a locally increased expression of neurotoxic tumour necrosis factor-alpha (TNF-alpha) and a locally decreased expression of neurotrophic epidermal growth factor (EGF). These recent findings suggest that cobalamin oppositely regulates the expression of TNF-alpha and EGF, and raise the possibility that these effects might be independent of its coenzyme function. Furthermore, adult Cbl-D patients have high levels of TNF-alpha and low levels of EGF in the serum and cerebrospinal fluid. Serum levels of TNF-alpha and EGF of cobalamin-treated patients normalize concomitantly with haematological disease remission. These observations suggest that cobalamin deficiency induces an imbalance in TNF-alpha and EGF levels in biological fluids that might have a role in the pathogenesis of the damage caused by pernicious anaemia.     

Epidermal Growth Factor in the CNS: A Beguiling Journey from Integrated Cell Biology to Multiple Sclerosis. An Extensive Translational Overview

This article reviews the wealth of papers dealing with the different effects of epidermal growth factor (EGF) on oligodendrocytes, astrocytes, neurons, and neural stem cells (NSCs). EGF induces the in vitro and in vivo proliferation of NSCs, their migration, and their differentiation towards the neuroglial cell line. It interacts with extracellular matrix components. NSCs are distributed in different CNS areas, serve as a reservoir of multipotent cells, and may be increased during CNS demyelinating diseases. EGF has pleiotropic differentiative and proliferative effects on the main CNS cell types, particularly oligodendrocytes and their precursors, and astrocytes. EGF mediates the in vivo myelinotrophic effect of cobalamin on the CNS, and modulates the synthesis and levels of CNS normal prions (PrP^Cs), both of which are indispensable for myelinogenesis and myelin maintenance. EGF levels are significantly lower in the cerebrospinal fluid and spinal cord of patients with multiple sclerosis (MS), which probably explains remyelination failure, also because of the EGF marginal role in immunology. When repeatedly administered, EGF protects mouse spinal cord from demyelination in various experimental models of autoimmune encephalomyelitis. It would be worth further investigating the role of EGF in the pathogenesis of MS because of its multifarious effects.

     

Prion protein with a mutant N-terminal octarepeat region undergoes cobalamin-dependent assembly into high–molecular weight complexes

The cellular prion protein (PrP^C) has a C-terminal globular domain and a disordered N-terminal region encompassing five octarepeats (ORs). Encounters between Cu(II) ions and four OR sites produce interchangeable binding geometries; however, the significance of Cu(II) binding to ORs in different combinations is unclear. To understand the impact of specific binding geometries, OR variants were designed that interact with multiple or single Cu(II) ions in specific locked coordinations. Unexpectedly, we found that one mutant produced detergent-insoluble, protease-resistant species in cells in the absence of exposure to the infectious prion protein isoform, scrapie-associated prion protein (PrP^Sc). Formation of these assemblies, visible as puncta, was reversible and dependent upon medium formulation. Cobalamin (Cbl), a dietary cofactor containing a corrin ring that coordinates a Co³⁺ ion, was identified as a key medium component, and its effect was validated by reconstitution experiments. Although we failed to find evidence that Cbl interacts with Cu-binding OR regions, we instead noted interactions of Cbl with the PrP^C C-terminal domain. We found that some interactions occurred at a binding site of planar tetrapyrrole compounds on the isolated globular domain, but others did not, and N-terminal sequences additionally had a marked effect on their presence and position. Our studies define a conditional effect of Cbl wherein a mutant OR region can act in cis to destabilize a globular domain with a wild type sequence. The unexpected intersection between the properties of PrP^Sc''s disordered region, Cbl, and conformational remodeling events may have implications for understanding sporadic prion disease that does not involve exposure to PrP^Sc.     

Role of Prion protein-EGFR multimolecular complex during neuronal differentiation of human dental pulp-derived stem cells

Cellular prion protein (PrP^C) is expressed in a wide variety of stem cells in which regulates their self-renewal as well as differentiation potential. In this study we investigated the presence of PrP^C in human dental pulp-derived stem cells (hDPSCs) and its role in neuronal differentiation process. We show that hDPSCs expresses early PrP^C at low concentration and its expression increases after two weeks of treatment with EGF/bFGF. Then, we analyzed the association of PrP^C with gangliosides and EGF receptor (EGF-R) during neuronal differentiation process. PrP^C associates constitutively with GM2 in control hDPSCs and with GD3 only after neuronal differentiation. Otherwise, EGF-R associates weakly in control hDPSCs and more markedly after neuronal differentiation.

To analyze the functional role of PrP^C in the signal pathway mediated by EGF/EGF-R, a siRNA PrP was applied to ablate PrP^C and its function. The treatment with siRNA PrP significantly prevented Akt and ERK1/2 phosphorylation induced by EGF.

Moreover, siRNA PrP treatment significantly prevented neuronal-specific antigens expression induced by EGF/bFGF, indicating that cellular prion protein is essential for EGF/bFGF-induced hDPSCs differentiation.

These results suggest that PrP^C interact with EGF-R within lipid rafts, playing a role in the multimolecular signaling complexes involved in hDPSCs neuronal differentiation.     

Sialylation of Prion Protein Controls the Rate of Prion Amplification,the Cross-Species Barrier,the Ratio of PrPSc Glycoform and Prion Infectivity

The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrP^C) into the disease-associated, transmissible form (PrP^Sc). PrP^C is a sialoglycoprotein that contains two conserved N-glycosylation sites. Among the key parameters that control prion replication identified over the years are amino acid sequence of host PrP^C and the strain-specific structure of PrP^Sc. The current work highlights the previously unappreciated role of sialylation of PrP^C glycans in prion pathogenesis, including its role in controlling prion replication rate, infectivity, cross-species barrier and PrP^Sc glycoform ratio. The current study demonstrates that undersialylated PrP^C is selected during prion amplification in Protein Misfolding Cyclic Amplification (PMCAb) at the expense of oversialylated PrP^C. As a result, PMCAb-derived PrP^Sc was less sialylated than brain-derived PrP^Sc. A decrease in PrP^Sc sialylation correlated with a drop in infectivity of PMCAb-derived material. Nevertheless, enzymatic de-sialylation of PrP^C using sialidase was found to increase the rate of PrP^Sc amplification in PMCAb from 10- to 10,000-fold in a strain-dependent manner. Moreover, de-sialylation of PrP^C reduced or eliminated a species barrier of for prion amplification in PMCAb. These results suggest that the negative charge of sialic acid controls the energy barrier of homologous and heterologous prion replication. Surprisingly, the sialylation status of PrP^C was also found to control PrP^Sc glycoform ratio. A decrease in PrP^C sialylation levels resulted in a higher percentage of the diglycosylated glycoform in PrP^Sc. 2D analysis of charge distribution revealed that the sialylation status of brain-derived PrP^C differed from that of spleen-derived PrP^C. Knocking out lysosomal sialidase Neu1 did not change the sialylation status of brain-derived PrP^C, suggesting that Neu1 is not responsible for desialylation of PrP^C. The current work highlights previously unappreciated role of PrP^C sialylation in prion diseases and opens multiple new research directions, including development of new therapeutic approaches.     

PrP mRNA and protein expression in brain and PrPc in CSF in Creutzfeldt-Jakob disease MM1 and VV2

Creutzfeldt-Jakob disease (CJD) is a heterogenic neurodegenerative disorder associated with abnormal post-translational processing of cellular prion protein (PrP^c). CJD displays distinctive clinical and pathological features which correlate with the genotype at the codon 129 (methionine or valine: M or V respectively) in the prion protein gene and with size of the protease-resistant core of the abnormal prion protein PrP^sc (type 1: 20/21 kDa and type 2: 19 kDa). MM1 and VV2 are the most common sporadic CJD (sCJD) subtypes. PrP mRNA expression levels in the frontal cortex and cerebellum are reduced in sCJD in a form subtype-dependent. Total PrP protein levels and PrP^sc levels in the frontal cortex and cerebellum accumulate differentially in sCJD MM1 and sCJD VV2 with no relation between PrP^sc deposition and spongiform degeneration and neuron loss, but with microgliosis, and IL6 and TNF-α response. In the CSF, reduced PrP^c, the only form present in this compartment, occurs in sCJD MM1 and VV2. PrP mRNA expression is also reduced in the frontal cortex in advanced stages of Alzheimer disease, Lewy body disease, progressive supranuclear palsy, and frontotemporal lobe degeneration, but PrP^c levels in brain varies from one disease to another. Reduced PrP^c levels in CSF correlate with PrP mRNA expression in brain, which in turn reflects severity of degeneration in sCJD.     

Comparison of the Anti-Prion Mechanism of Four Different Anti-Prion Compounds,Anti-PrP Monoclonal Antibody 44B1, Pentosan Polysulfate,Chlorpromazine, and U18666A,in Prion-Infected Mouse Neuroblastoma Cells

Molecules that inhibit the formation of an abnormal isoform of prion protein (PrP^Sc) in prion-infected cells are candidate therapeutic agents for prion diseases. Understanding how these molecules inhibit PrP^Sc formation provides logical basis for proper evaluation of their therapeutic potential. In this study, we extensively analyzed the effects of the anti-PrP monoclonal antibody (mAb) 44B1, pentosan polysulfate (PPS), chlorpromazine (CPZ) and U18666A on the intracellular dynamics of a cellular isoform of prion protein (PrP^C) and PrP^Sc in prion-infected mouse neuroblastoma cells to re-evaluate the effects of those agents. MAb 44B1 and PPS rapidly reduced PrP^Sc levels without altering intracellular distribution of PrP^Sc. PPS did not change the distribution and levels of PrP^C, whereas mAb 44B1 appeared to inhibit the trafficking of cell surface PrP^C to organelles in the endocytic-recycling pathway that are thought to be one of the sites for PrP^Sc formation. In contrast, CPZ and U18666A initiated the redistribution of PrP^Sc from organelles in the endocytic-recycling pathway to late endosomes/lysosomes without apparent changes in the distribution of PrP^C. The inhibition of lysosomal function by monensin or bafilomycin A1 after the occurrence of PrP^Sc redistribution by CPZ or U18666A partly antagonized PrP^Sc degradation, suggesting that the transfer of PrP^Sc to late endosomes/lysosomes, possibly via alteration of the membrane trafficking machinery of cells, leads to PrP^Sc degradation. This study revealed that precise analysis of the intracellular dynamics of PrP^C and PrP^Sc provides important information for understanding the mechanism of anti-prion agents.     

Prion Protein Modulates Cellular Iron Uptake: A Novel Function with Implications for Prion Disease Pathogenesis

Converging evidence leaves little doubt that a change in the conformation of prion protein (PrP^C) from a mainly α-helical to a β-sheet rich PrP-scrapie (PrP^Sc) form is the main event responsible for prion disease associated neurotoxicity. However, neither the mechanism of toxicity by PrP^Sc, nor the normal function of PrP^C is entirely clear. Recent reports suggest that imbalance of iron homeostasis is a common feature of prion infected cells and mouse models, implicating redox-iron in prion disease pathogenesis. In this report, we provide evidence that PrP^C mediates cellular iron uptake and transport, and mutant PrP forms alter cellular iron levels differentially. Using human neuroblastoma cells as models, we demonstrate that over-expression of PrP^C increases intra-cellular iron relative to non-transfected controls as indicated by an increase in total cellular iron, the cellular labile iron pool (LIP), and iron content of ferritin. As a result, the levels of iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) are decreased, and expression of iron storage protein ferritin is increased. The positive effect of PrP^C on ferritin iron content is enhanced by stimulating PrP^C endocytosis, and reversed by cross-linking PrP^C on the plasma membrane. Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP^102L decreases ferritin iron content significantly relative to PrP^C expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation. Neither PrP^C nor the mutant PrP forms influence the rate or amount of iron released into the medium, suggesting a functional role for PrP^C in cellular iron uptake and transport to ferritin, and dysfunction of PrP^C as a significant contributing factor of brain iron imbalance in prion disorders.     

High levels of Cellular Prion Protein improve astrocyte development

Prion protein (PrP^C) has neuroprotective functions and herein we demonstrate that astrocytes from PrP^C-over-expressing mice are more resistant to induced cell death than wild-type astrocytes. The Stress-Inducible-Protein 1 (STI1), a PrP^C ligand, prevents cell death in both wild-type and PrP^C-over-expressing astrocytes through the activation of protein-kinase-A. Cultured embryonic astrocytes and brain extracts from PrP^C-over-expressing mice show higher glial fibrillary acidic protein expression and reduced vimentin and nestin levels when compared to wild-type astrocytes, suggesting faster astrocyte maturation in the former mice. Our data indicate that PrP^C levels modulate astrocyte development, and that PrP^C–STI1 interaction contributes to protect against astrocyte death.     

Quantitative and qualitative analysis of cellular prion protein (PrPC) expression in bovine somatic tissues

The host encoded cellular prion protein (PrP^C) is an N-linked glycoprotein tethered to the cell membrane by a glycophosphatidylinositol (GPI) anchor. Under certain conditions, PrP^C can undergo conversion into a conformationally-altered isoform (PrP^Sc) widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Understanding the tissue-specific expression of PrP^C is crucial considering that cells expressing high levels of PrP^C bear a risk for conversion and accumulation of PrP^Sc. In the present study, fifteen bovine somatic tissues were analyzed for PrP^C expression by quantitative western blot and immunohistochemistry. Quantitative western blot analysis revealed highest expression of PrP^C in cerebellum, obex and spinal cord. Intermediate levels were detected in thymus, intestine, nerve, heart and spleen, and lower levels in lung, muscle, kidney, lymph node, skin, pancreas and liver. Immunohistochemical analysis detected intense cellular-specific PrP^C staining in neurons, thymocytes and lymphocytes. PrP^C was also detected in the enteric wall, pancreatic islets of langerhans, myocardium, pulmonary alveolar sacs, renal glomeruli and dermal epithelial cells. This study demonstrated the quantitatively varied, wide-spread, tissue- and cell-specific expression pattern of PrP^C in bovine somatic tissues. The importance of this study is to lay the foundation for understanding the tissue-specific expression of PrP^C and to consider the potential participation of more bovine tissues in the transmission of BSE infection.Key words: cellular prion protein (PrP^C), protein expression, bovine somatic tissues, BSE, western blot, immunohistochemistry     

CSF prion protein concentration and cognition in patients with Alzheimer disease

Background/Objective: PrP^c has been suggested to play a role in AD pathophysiology. CSF concentrations of PrP^c have been shown to be reduced in AD compared with healthy controls. Furthermore, serum levels of PrP^c have recently been reported to be associated with the cognitive status of healthy elderly subjects. Therefore, we hypothesized that CSF levels of PrP^c could be associated with cognitive function of AD patients at the time of diagnosis.

Methods: AD patients (n = 114) included into an observational study underwent CERAD testing and lumbar puncture at time of diagnosis / study inclusion. CSF PrP^c was determined. Generalized linear models were fitted to assess the associations of PrP^c plus a variety of possible confounding factors and CERAD subscale measures.

Results: No association of CSF PrP^c and cognitive status could be established, while other factors (i.e., use of antipsychotic drugs, use of anti-dementia drugs, female sex, pre-progression time) were related to worse cognitive function in some domains.

Conclusion: CSF PrP^c appears not to be a useful biochemical surrogate of cognitive status in AD at the time of diagnosis. Follow-up analyses will examine possible associations with the speed of cognitive decline.     

Octapeptide repeat region of prion protein (PrP) is required at an early stage for production of abnormal prion protein in PrP-deficient neuronal cell line

An abnormal isoform of prion protein (PrP^Sc), which is composed of the same amino acids as cellular PrP (PrP^C) and has proteinase K (PK)-resistance, hypothetically converts PrP^C into PrP^Sc. To investigate the region important for PrP^Sc production, we examined the levels of PrP^Sc in PrP gene-deficient cells (HpL3-4) expressing PrP^C deleted of various regions including the octapeptide repeat region (OR) or hydrophobic region (HR). After Chandler or Obihiro prion infection, PrP^Sc was produced in HpL3-4 cells expressing wild-type PrP^C or PrP^C deleted of HR at an early stage and further reduced to below the detectable level, whereas cells expressing PrP^C deleted of OR showed no PrP^Sc production. The results suggest that OR of PrP^C is required for the early step of efficient PrP^Sc production.     

Maternal deficiency of protein or protein and calories during lactation: effect upon CNS myelin subfraction formation in rat offspring.

The present study has examined the effects of maternal protein and protein-calorie deficiency during lactation on the development of CNS myelin subfractions in rat offspring. The offspring of both the protein and protein-calorie deficient rats had decreased brain and body weights, as well as delayed CNS myelination. Delayed active CNS myelination was demonstrated by the fact that 53-day-old nutritionally stressed pups incorporated significantly more [³H]leucine and [¹⁴C]glucose into all myelin subfractions than age-matched controls. Delayed myelination was also supported by the tremendous accretion of myelin proteins in the nutritionally deprived pups between 25 and 53 days of age. Despite the delayed active synthesis of myelin, the myelin deficit persisted in the offspring of protein deficient rats. These offspring had a deficiency of light + medium myelin throughout the study. At 17 days, both groups of nutritionally stressed rats had an excess of the high molecular weight proteins in heavy myelin. Heavy myelin from 17 day offspring of protein-calorie deficient rats had a deficiency of basic proteins, while that from the offspring of protein deficient rats had a deficiency of proteolipid protein. The protein composition of all myelin subfractions was normal at 53 days.     

The Cellular Prion Protein Interacts with the Tissue Non-Specific Alkaline Phosphatase in Membrane Microdomains of Bioaminergic Neuronal Cells

Background

The cellular prion protein, PrP^C, is GPI anchored and abundant in lipid rafts. The absolute requirement of PrP^C in neurodegeneration associated to prion diseases is well established. However, the function of this ubiquitous protein is still puzzling. Our previous work using the 1C11 neuronal model, provided evidence that PrP^C acts as a cell surface receptor. Besides a ubiquitous signaling function of PrP^C, we have described a neuronal specificity pointing to a role of PrP^C in neuronal homeostasis. 1C11 cells, upon appropriate induction, engage into neuronal differentiation programs, giving rise either to serotonergic (1C11^5-HT) or noradrenergic (1C11^NE) derivatives.

Methodology/Principal Findings

The neuronal specificity of PrP^C signaling prompted us to search for PrP^C partners in 1C11-derived bioaminergic neuronal cells. We show here by immunoprecipitation an association of PrP^C with an 80 kDa protein identified by mass spectrometry as the tissue non-specific alkaline phosphatase (TNAP). This interaction occurs in lipid rafts and is restricted to 1C11-derived neuronal progenies. Our data indicate that TNAP is implemented during the differentiation programs of 1C11^5-HT and 1C11^NE cells and is active at their cell surface. Noteworthy, TNAP may contribute to the regulation of serotonin or catecholamine synthesis in 1C11^5-HT and 1C11^NE bioaminergic cells by controlling pyridoxal phosphate levels. Finally, TNAP activity is shown to modulate the phosphorylation status of laminin and thereby its interaction with PrP.

Conclusion/Significance

The identification of a novel PrP^C partner in lipid rafts of neuronal cells favors the idea of a role of PrP in multiple functions. Because PrP^C and laminin functionally interact to support neuronal differentiation and memory consolidation, our findings introduce TNAP as a functional protagonist in the PrP^C-laminin interplay. The partnership between TNAP and PrP^C in neuronal cells may provide new clues as to the neurospecificity of PrP^C function.     

Phosphatidylinositol-Glycan-Phospholipase D Is Involved in Neurodegeneration in Prion Disease

PrP^Sc is formed from a normal glycosylphosphatidylinositol (GPI)-anchored prion protein (PrP^C) by a posttranslational modification. Most GPI-anchored proteins have been shown to be cleaved by GPI phospholipases. Recently, GPI-phospholipase D (GPI-PLD) was shown to be a strictly specific enzyme for GPI anchors. To investigate the involvement of GPI-PLD in the processes of neurodegeneration in prion diseases, we examined the mRNA and protein expression levels of GPI-PLD in the brains of a prion animal model (scrapie), and in both the brains and cerebrospinal fluids (CSF) of sporadic and familial Creutzfeldt-Jakob disease (CJD) patients. We found that compared with controls, the expression of GPI-PLD was dramatically down-regulated in the brains of scrapie-infected mice, especially in the caveolin-enriched membrane fractions. Interestingly, the observed decrease in GPI-PLD expression levels began at the same time that PrP^Sc began to accumulate in the infected brains and this decrease was also observed in both the brain and CSF of CJD patients; however, no differences in expression were observed in either the brains or CSF specimens from Alzheimer’s disease patients. Taken together, these results suggest that the down-regulation of GPI-PLD protein may be involved in prion propagation in the brains of prion diseases.     

Role of α7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrP^C) is a conserved glycosylphosphatidylinositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP^C extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrP^C-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP^C engagement induces an increase in intracellular Ca²⁺ levels. This effect was not detected in PrP^C-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP^C. Using a best candidate approach to test for potential channels involved in Ca²⁺ influx evoked by STI1-PrP^C, we found that α-bungarotoxin, a specific inhibitor for α7 nicotinic acetylcholine receptor (α7nAChR), was able to block PrP^C-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when α7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP^C and allowed reconstitution of signaling by PrP^C-STI1 interaction. These results indicate that STI1 can interact with the PrP^C·α7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.