Alessandro Bertoli

Protein misfolding in Alzheimer's and Parkinson's disease: Genetics and molecular mechanisms

The accumulation of altered proteins is a common pathogenic mechanism in several neurodegenerative disorders. A causal role of protein aggregation was originally proposed in Alzheimers disease (AD) where extracellular deposition of beta-amyloid (Abeta) is the main neuropathological feature. It is now believed that intracellular deposition of aggregated proteins may be relevant in Parkinsons disease (PD), amyotrophic lateral sclerosis and polyglutamine disorders. An impairment of ubiquitin-proteasome system (UPS) appears directly involved in these disorders. We reviewed the results on the role of protein misfolding in AD and PD and the influence of mutations associated with these diseases on the expression of amyloidogenic proteins. Results of genetic screening of familial cases of AD and PD are summarized. In the familial AD population (70 subjects) we found several mutations of the presenilin 1 (PS1) gene with a frequency of 12.8% and one mutation in the gene encoding the protein precursor of amyloid (APP) (1.4%). One mutation of Parkin in the homozygous form and two in the heterozygous form were identified in our PD population. We also reported data obtained with synthetic peptides and other experimental models, for evaluation of the pathogenic role of mutations in terms of protein misfolding.

The metabolism and imaging in live cells of the bovine prion protein in its native form or carrying single amino acid substitutions.

Prion diseases are probably caused by an abnormal form of a cellular glycoprotein, the prion protein. Recent evidence suggests that the prion strain causing BSE has been transmitted to humans, thereby provoking a variant form of Creutzfeldt-Jacob disease. In this work, we analyzed the behavior of normal and malformed isoforms of the bovine PrP in transfected mammalian cell lines. Biochemical and immunocytochemical assays were complimented with imaging of live cells expressing fusion constructs between PrP and GFP. Bovine homologues of human E200K and D178N (129M) mutations were used as models of pathogenic isoforms. We show that the GFP does not impair the metabolism of native and mutant bPrPs and is thus a valid marker of PrP cellular distribution. We also show that each amino acid replacement provokes alterations in the cell sorting and processing of bPrP. These are different from those ascribed to both murine mutant homologues. However, human and bovine PrPs carrying the D178N genotype had similar cellular behavior.

Proteasome inhibition and aggregation in Parkinson's disease: a comparative study in untransfected and transfected cells

Dysfunction of the ubiquitin‐proteasome system (UPS) has been implicated in Parkinsons disease (PD) and other neurodegenerative disorders. We have investigated the effect of UPS inhibition on the metabolism of α‐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.

Cellular Prion Protein Promotes Regeneration of Adult Muscle Tissue

ABSTRACT It is now well established that the conversion of the cellular prion protein, PrPC, into its anomalous conformer, PrPSc, is central to the onset of prion disease. However, both the mechanism of prion-related neurodegeneration and the physiologic role of PrPC are still unknown. The use of animal and cell models has suggested a number of putative functions for the protein, including cell signaling, adhesion, proliferation, and differentiation. Given that skeletal muscles express significant amounts of PrPC and have been related to PrPC pathophysiology, in the present study, we used skeletal muscles to analyze whether the protein plays a role in adult morphogenesis. We employed an in vivo paradigm that allowed us to compare the regeneration of acutely damaged hind-limb tibialis anterior muscles of mice expressing, or not expressing, PrPC. Using morphometric and biochemical parameters, we provide compelling evidence that the absence of PrPC significantly slows the regeneration process compared to wild-type muscles by attenuating the stress-activated p38 pathway, and the consequent exit from the cell cycle, of myogenic precursor cells. Demonstrating the specificity of this finding, restoring PrPC expression completely rescued the muscle phenotype evidenced in the absence of PrPC.

Cellular prion protein is implicated in the regulation of local Ca2+ movements in cerebellar granule neurons

J. Neurochem. (2011) 116, 881–890.

Is, indeed, the prion protein a Harlequin servant of "many" masters?

Tens of putative interacting partners of the cellular prion protein (PrPC) have been identified, yet the physiologic role of PrPC remains unclear. For the first time, however, a recent paper has demonstrated that the absence of PrPC produces a lethal phenotype. Starting from this evidence, here we discuss the validity of past and more recent literature supporting that, as part of protein platforms at the cell surface, PrPC may bridge extracellular matrix molecules and membrane proteins to intracellular signaling pathways.

From cell protection to death: May Ca2+ signals explain the chameleonic attributes of the mammalian prion protein?

It is now accepted that a conformational change of the cellular prion protein (PrP(C)) generates the prion, the infectious agent responsible for lethal neurodegenerative disorders, named transmissible spongiform encephalopathies, or prion diseases. The mechanisms of prion-associated neurodegeneration are still obscure, as is the cell role of PrP(C), although increasing evidence attributes to PrP(C) important functions in cell survival. Such a behavioral dichotomy thus enables the prion protein to switch from a benign role under normal conditions, to the execution of neurons during disease. By reviewing data from models of prion disease and PrP(C)-null paradigms, which suggest a relation between the prion protein and Ca(2+) homeostasis, here we discuss the possibility that Ca(2+) is the factor behind the enigma of the pathophysiology of PrP(C). Ca(2+) features in almost all processes of cell signaling, and may thus tell us much about a protein that pivots between health and disease.

Possible role for Ca2+ in the pathophysiology of the prion protein?

Transmissible spongiform encephalopathies, or prion diseases, are lethal neurodegenerative disorders caused by the infectious agent named prion, whose main constituent is an aberrant conformational isoform of the cellular prion protein, PrP(C) . The mechanisms of prion-associated neurodegeneration and the physiologic function of PrP(C) are still unclear, although it is now increasingly acknowledged that PrP(C) plays a role in cell differentiation and survival. PrP(C) thus exhibits dichotomic attributes, as it can switch from a benign function under normal conditions to the triggering of neuronal death during disease. By reviewing data from models of prion infection and PrP-knockout paradigms, here we discuss the possibility that Ca(2+) is the hidden factor behind the multifaceted behavior of PrP(C) . By featuring in almost all processes of cell signaling, Ca(2+) might explain diverse aspects of PrP(C) pathophysiology, including the recently proposed one in which PrP(C) acts as a mediator of synaptic degeneration in Alzheimers disease.

Relative quantification of membrane proteins in wild-type and prion protein (PrP)-knockout cerebellar granule neurons.

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.

The prion protein constitutively controls neuronal store-operated Ca2+ entry through Fyn kinase

The prion protein (PrPC) is a cell surface glycoprotein mainly expressed in neurons, whose misfolded isoforms generate the prion responsible for incurable neurodegenerative disorders. Whereas PrPC involvement in prion propagation is well established, PrPC physiological function is still enigmatic despite suggestions that it could act in cell signal transduction by modulating phosphorylation cascades and Ca2+ homeostasis. Because PrPC binds neurotoxic protein aggregates with high-affinity, it has also been proposed that PrPC acts as receptor for amyloid-β (Aβ) oligomers associated with Alzheimer’s disease (AD), and that PrPC-Aβ binding mediates AD-related synaptic dysfunctions following activation of the tyrosine kinase Fyn. Here, use of gene-encoded Ca2+ probes targeting different cell domains in primary cerebellar granule neurons (CGN) expressing, or not, PrPC, allowed us to investigate whether PrPC regulates store-operated Ca2+ entry (SOCE) and the implication of Fyn in this control. Our findings show that PrPC attenuates SOCE, and Ca2+ accumulation in the cytosol and mitochondria, by constitutively restraining Fyn activation and tyrosine phosphorylation of STIM1, a key molecular component of SOCE. This data establishes the existence of a PrPC-Fyn-SOCE triad in neurons. We also demonstrate that treating cerebellar granule and cortical neurons with soluble Aβ(1–42) oligomers abrogates the control of PrPC over Fyn and SOCE, suggesting a PrPC-dependent mechanizm for Aβ-induced neuronal Ca2+ dyshomeostasis.