Papers by Alessandro Bertoli
The International Journal of Biochemistry & Cell Biology, Oct 1, 2004
Doppel is the first identified homologue of the prion protein (PrPc) implicated in prion disease.... more Doppel is the first identified homologue of the prion protein (PrPc) implicated in prion disease. Doppel is considered an N-truncated form of PrPc, and shares with PrPc several structural and biochemical features. When over expressed in the brain of some PrP knockout animals, it provokes cerebellar ataxia. As this phenotype is rescued by reintroducing the PrP gene, it has been suggested that Doppel and PrPc have antagonistic functions and may compete for a common ligand. However, a direct interaction between the two proteins has recently been observed. To investigate whether the neuronal environment is suitable for such possibility, human Doppel and PrPc were expressed separately, or together, in neuroblastoma cells, and then studied by biochemical and immunomicroscopic tools, as well as in intact cells expressing fluorescent fusion constructs. The results demonstrate that Doppel and PrPc co-patch extensively at the plasma membrane, and get internalized together after ganglioside cross-linking by cholera toxin or addition of an antibody against only one of the proteins. These processes no longer occur if the integrity of rafts is disrupted. We also show that, whereas each protein expressed alone occupies Triton X-100-insoluble membrane microdomains, co-transfected Doppel and PrPc redistribute together into a less ordered lipidic environment. All these features are consistent with interactions occurring between Doppel and PrPc in our neuronal cell model.
Biochemical Society Transactions, Oct 1, 2000
Neurodegenerative diseases like Creutzfeldt-Jacob in humans or transmissible spongiform encephalo... more Neurodegenerative diseases like Creutzfeldt-Jacob in humans or transmissible spongiform encephalopathy in animals are attributed to conformational conversion of the GPI-anchored cellular prion protein (PrPc) into a pathogenic isoform. Despite the abundance of PrPc in the central nervous system, its normal function is unknown. Recently, our group has demonstrated that PrPc is a specific, highaffinity receptor for laminin, the most important non collagenic extracellular matrix component which plays a fundamental role in neuronal differentiation. The binding site resides in a carboxi-terminal decapeptide (RNIAEIIKDI) from they1 laminin chain (Brain Res Mol Brain Res 76: 85-92,2000). Moreover, we have previously
International Journal of Molecular Sciences, Nov 1, 2021
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Journal of Neurochemistry, Dec 23, 2003
Dysfunction of the ubiquitin-proteasome system (UPS) has been implicated in Parkinson's disease (... more Dysfunction of the ubiquitin-proteasome system (UPS) has been implicated in Parkinson's disease (PD) and other neurodegenerative disorders. We have investigated the effect of UPS inhibition on the metabolism of a-synuclein (SYN) and parkin, two proteins genetically and histopathologically associated to PD. Pharmacological inhibition of proteasome induced accumulation of both parkin and SYN in transfected PC12 cells. We found that this effect was caused by increased protein synthesis rather than impairment of protein degradation, suggesting that inhibition of the UPS might lead to non-specific up-regulation of cytomegalovirus (CMV)-driven transcription. To investigate whether endogenous parkin and SYN can be substrate of the UPS, untransfected PC12 cells and primary mesencephalic neurones were exposed to proteasome inhibitors, and parkin and SYN expression was evaluated at both protein and mRNA level. Under these conditions, we found that proteasome inhibitors did not affect the level of endogenous parkin and SYN. However, we confirmed that dopaminergic neurones were selectively vulnerable to the toxicity of proteasome inhibitors. Our results indicate that studies involving the use of proteasome inhibitors, particularly those in which proteins are expressed from a heterologous promoter, are subjected to potential artefacts that need to be considered for the interpretation of the role of UPS in PD pathogenesis.
International Journal of Molecular Sciences, Jun 29, 2021
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Chemical Senses, Jun 16, 2011
A conformational conversion of the cellular prion protein (PrP C) is now recognized as the causal... more A conformational conversion of the cellular prion protein (PrP C) is now recognized as the causal event of fatal neurodegenerative disorders, known as prion diseases. In spite of long-lasting efforts, however, the physiological role of PrP C remains unclear. It has been reported that PrP C is expressed in various areas of the olfactory system, including the olfactory epithelium, but its precise localization in olfactory sensory neurons (OSNs) is still debated. Here, using immunohistochemistry tools, we have reinvestigated the expression and localization of PrP C in the olfactory epithelium of adult congenic mice expressing different PrP C amounts, that is, wild-type, PrP-knockout, and transgenic PrP C-overexpressing animals. We found that PrP C was expressed in OSNs, in which, however, it was unevenly distributed, being detectable at low levels in cell bodies, dendrites and apical layer, and more abundantly in axons. We also studied the involvement of PrP C in the response of the olfactory epithelium to odorants, by comparing the electro-olfactograms of the 3 mouse lines subjected to different stimulation protocols. We found no significant difference between the 3 PrP genotypes, supporting previous reports that exclude a direct action of PrP C in the early signal transduction activity of the olfactory epithelium.
International Journal of Molecular Sciences, Oct 20, 2020
Prion diseases are rare transmissible neurodegenerative disorders caused by the accumulation of a... more Prion diseases are rare transmissible neurodegenerative disorders caused by the accumulation of a misfolded isoform (PrP Sc) of the cellular prion protein (PrP C) in the central nervous system (CNS). Neuropathological hallmarks of prion diseases are neuronal loss, astrogliosis, and enhanced microglial proliferation and activation. As immune cells of the CNS, microglia participate both in the maintenance of the normal brain physiology and in driving the neuroinflammatory response to acute or chronic (e.g., neurodegenerative disorders) insults. Microglia involvement in prion diseases, however, is far from being clearly understood. During this review, we summarize and discuss controversial findings, both in patient and animal models, suggesting a neuroprotective role of microglia in prion disease pathogenesis and progression, or-conversely-a microglia-mediated exacerbation of neurotoxicity in later stages of disease. We also will consider the active participation of PrP C in microglial functions, by discussing previous reports, but also by presenting unpublished results that support a role for PrP C in cytokine secretion by activated primary microglia.
Journal of Proteome Research, Nov 18, 2011
Approximately 25% of eukaryotic proteins possessing homology to at least two transmembrane domain... more Approximately 25% of eukaryotic proteins possessing homology to at least two transmembrane domains are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is not usually well represented in proteome-wide experiments due to their refractory nature. Here we present a quantitative mass spectrometry-based comparison of membrane protein expression in cerebellar granule neurons grown in primary culture that were isolated from wild-type mice and mice lacking the cellular prion protein. This protein is a cell-surface glycoprotein that is mainly expressed in the central nervous system and is involved in several neurodegenerative disorders, though its physiological role is unclear. We used a low specificity enzyme α-chymotrypsin to digest membrane proteins preparations that had been separated by SDS-PAGE. The resulting peptides were labeled with tandem mass tags and analyzed by MS. The differentially expressed proteins identified using this approach were further analyzed by multiple reaction monitoring to confirm the expression level changes.
European Biophysics Journal, Dec 1, 1994
We studied the activation properties of members of the Shaker-related subfamily of voltage-gated ... more We studied the activation properties of members of the Shaker-related subfamily of voltage-gated K+ channels cloned from rat brain and expressed in Xenopus oocytes. We find that Kv1.1, Kv1.4, Kv1.5, and Kv1.6 have similar activation and deactivation kinetics. The k+ currents produced by step depolarisations increase with a sigmoidal time course that can be described by a delay and by the derivative of the current at the inflection point. The delay tends to zero and the logarithmic derivative seems to approach a finite value at large positive voltages, but these asymptotic values are not yet reached at +80 mV. Deactivation of the currents upon stepping to negative membrane potentials below -60 mV is fairly well described by a single exponential. The decrease of the deactivation time constant at increasingly negative voltages tends to become less steep, indicating that this parameter also has a finite limiting value, which is not yet reached, however, at -160 mV. The various clones studied have very similar voltage dependencies of activation with half-activation voltages ranging between -50 and -11 mV and maximum steepness yielding and e-fold change for voltage increments between 3.8 and 7.0 mV. The shallower activation curve of Kv1.4 is likely to be due to coupling with the fast inactivation process present in this clone.
Biochemical Journal, Nov 7, 2000
On the basis of far-Western blot and plasmon resonance (BIAcore) experiments, we show here that r... more On the basis of far-Western blot and plasmon resonance (BIAcore) experiments, we show here that recombinant bovine prion protein (bPrP) (25-242) strongly interacts with the catalytic α\αh subunits of protein kinase CK2 (also termed ' casein kinase 2 '). This association leads to increased phosphotransferase activity of CK2α, tested on calmodulin or specific peptides as substrate. We also show that bPrP counteracts the inhibition of calmodulin phosphorylation promoted by the regulatory β subunits of CK2. A truncated form of bPrP encompassing the Cterminal domain (residues 105-242) interacts with CK2 but does Abbreviations used : bPrP, bovine PrP ; CK1, casein kinase 1 ; CK2, ' casein kinase 2 ' ; I-2, inhibitor 2 of protein phosphatase 1 ; PrP, prion protein ; PrP c , cellular PrP; PrP Sc , pathological isoform of PrP ; SPR, surface plasmon resonance. 1 To whom correspondence should be addressed (e-mail pinna!civ.bio.unipd.it). not affect its catalytic activity. The opposite is found with the Nterminal fragment of bPrP (residues 25-116), although the stimulation of catalysis is less efficient than with full-size bPrP. These results disclose the potential of the PrP to modulate the activity of CK2, a pleiotropic protein kinase that is particularly abundant in the brain.
Biochemical and Biophysical Research Communications, May 1, 2000
Ten protein kinases have been assayed for their ability to phosphorylate in vitro the recombinant... more Ten protein kinases have been assayed for their ability to phosphorylate in vitro the recombinant bovine PrP (25-242) (rbPrP). Substantial phosphorylation was observed with PKC, CK2, and two tyrosine kinases, Lyn and c-Fgr. With regard to CK2, phosphorylation occurs at Ser 154 with a stoichiometry of about 0.1 mol phosphate/mol rbPrP, which is doubled by mild heat treatment of rbPrP. Heat also reduces the overall protein ellipticity, suggesting that reversibly unfolded conformers are more susceptible to phosphorylation. Our data disclose the possibility that phosphorylation might modulate PrP biological activity.
evidenziato che il trattamento acuto dei neuroni con tali oligomeri altera la regolazione della P... more evidenziato che il trattamento acuto dei neuroni con tali oligomeri altera la regolazione della PrP C sul SOCE e su Fyn e l'ingresso di Ca 2+ nel mitocondrio a seguito dell'attivazione dei recettori ionotropici del glutammato. Questi dati suggeriscono pertanto l'esistenza di un meccanismo PrP C-dipendente che causa dis-omeostasi neuronale del Ca 2+ indotta dal peptide Aβ. X and, most remarkably, the capacity to self-propagate into host organisms through an auto-catalytic mechanism in which pre-formed PrP Sc promote the PrP C-PrP Sc conversion. Much data has been accumulated over the years to support the "protein only" hypothesis, including other "unorthodox" prion aspects. One of these is the observation that PrP Sc can give rise to different disease phenotypes that are faithfully propagated (Bruce and Fraser, 1991), which suggests the existence of prion strains distinguishable by hystopathological features, biochemical and physico-chemical properties, and by the incubation period of the disease. Recently, it was demonstrated modification in the circadian sleep rhythm (Collinge et al., 1994; Sakaguchi et al., 1996). The polypeptide coded by Prnp is subjected to several post-translational modifications: removal of the N-terminal signal peptide (aa 1-22), and of approximately 20 aa at the C-terminus (aa 231-253) to allow the GPI attachement (Stahl et al., 1990); the N-glycosilation at two asparagine residues (Asn181, 197) in the endoplasmic reticulum (ER); removal of mannose residues and addition of complex oligosaccharidic chains in the Golgi apparatus (Fig. 2). sheets (Fig. 3), which is further stabilized by a single disulfide bond (Riek et al., 1996; Zahn et al., 2000). The N-terminal contains five repetitions of eight aa (PHGGGWGQ) (octarepeats, OR) that can coordinate up-to six Cu 2+ (Brown et al., 1997). A hydrophobic region, located between the OR and the first α-helix (aa 106-126) is considered a possible trans-membrane domain, and exerts neurotoxic functions (Forloni et al., 1993). moiety. Like other GPI-anchored proteins, PrP C is located to sphingolipid-and cholesterol-abundant microdomains, known as detergent-resistant patches, or lipid rafts (Simons and Toomre, 2000), which many studies indicate as putative centres for signal transduction events. It remains to be tested whether the GPI-anchoring modulates other biological properties of PrP C , as shown for the fibroblast GPI-growth factor (Kohl et al., 2002). Sc (right). The α-helical and β-strand regions are shown in green and blue, respectively. It is to be noted the PrP Sc enrichement in the β-sheet content
Muscle & Nerve, Nov 26, 2015
Introduction: The cellular prion protein (PrP C) is commonly recognized as the precursor of prion... more Introduction: The cellular prion protein (PrP C) is commonly recognized as the precursor of prions, the infectious agents of the fatal transmissible spongiform encephalopathies, or prion diseases. Despite extensive effort, the physiological role of PrP C is still ambiguous. Evidence has suggested that PrP C is involved in different cellular functions, including peripheral nerve integrity and skeletal muscle physiology. Methods: We analyzed the age-dependent influence of PrP C on treadmill test-based aerobic exercise capacity and on a series of morphological and metabolic parameters using wild-type and genetically modified mice of different ages expressing, or knockout (KO) for, PrP C. Results: We found that aged PrP-KO mice displayed a reduction in treadmill performance compared with PrPexpressing animals, which was associated with peripheral nerve demyelination and alterations of skeletal muscle fiber type. Conclusion: PrP-KO mice have an age-dependent impairment of aerobic performance as a consequence of specific peripheral nerve and muscle alterations.
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Papers by Alessandro Bertoli