Mario Nizzari

Amyloid precursor protein and presenilin1 interact with the adaptor GRB2 and modulate ERK1,2 signaling

The amyloid precursor protein (APP) and the presenilins 1 and 2 are genetically linked to the development of familial Alzheimer disease. APP is a single-pass transmembrane protein and precursor of fibrillar and toxic amyloid-β peptides, which are considered responsible for Alzheimer disease neurodegeneration. Presenilins are multipass membrane proteins, involved in the enzymatic cleavage of APP and other signaling receptors and transducers. The role of APP and presenilins in Alzheimer disease development seems to be related to the formation of amyloid-β peptides; however, their physiological function, reciprocal interaction, and molecular mechanisms leading to neurodegeneration are unclear. APP and presenilins are also involved in multiple interactions with intracellular proteins, the significance of which is under investigation. Among the different APP-interacting proteins, we focused our interest on the GRB2 adaptor protein, which connects cell surface receptors to intracellular signaling pathways. In this study we provide evidence by co-immunoprecipitation experiments, confocal and electron microscopy, and by fluorescence resonance energy transfer experiments that both APP and presenilin1 interact with GRB2 in vesicular structures at the centrosome of the cell. The final target for these interactions is ERK1,2, which is activated in mitotic centrosomes in a PS1- and APP-dependent manner. These data suggest that both APP and presenilin1 can be part of a common signaling pathway that regulates ERK1,2 and the cell cycle.

An intronic ncRNA-dependent regulation of SORL1 expression affecting Aβ formation is upregulated in post-mortem Alzheimer's disease brain samples.

SUMMARY Recent studies indicated that sortilin-related receptor 1 (SORL1) is a risk gene for late-onset Alzheimers disease (AD), although its role in the aetiology and/or progression of this disorder is not fully understood. Here, we report the finding of a non-coding (nc) RNA (hereafter referred to as 51A) that maps in antisense configuration to intron 1 of the SORL1 gene. 51A expression drives a splicing shift of SORL1 from the synthesis of the canonical long protein variant A to an alternatively spliced protein form. This process, resulting in a decreased synthesis of SORL1 variant A, is associated with impaired processing of amyloid precursor protein (APP), leading to increased Aβ formation. Interestingly, we found that 51A is expressed in human brains, being frequently upregulated in cerebral cortices from individuals with Alzheimers disease. Altogether, these findings document a novel ncRNA-dependent regulatory pathway that might have relevant implications in neurodegeneration.

Peptide receptor targeting in cancer: the somatostatin paradigm.

Peptide receptors involved in pathophysiological processes represent promising therapeutic targets. Neuropeptide somatostatin (SST) is produced by specialized cells in a large number of human organs and tissues. SST primarily acts as inhibitor of endocrine and exocrine secretion via the activation of five G-protein-coupled receptors, named sst1–5, while in central nervous system, SST acts as a neurotransmitter/neuromodulator, regulating locomotory and cognitive functions. Critical points of SST/SST receptor biology, such as signaling pathways of individual receptor subtypes, homo- and heterodimerization, trafficking, and cross-talk with growth factor receptors, have been extensively studied, although functions associated with several pathological conditions, including cancer, are still not completely unraveled. Importantly, SST exerts antiproliferative and antiangiogenic effects on cancer cells in vitro, and on experimental tumors in vivo. Moreover, SST agonists are clinically effective as antitumor agents for pituitary adenomas and gastro-pancreatic neuroendocrine tumors. However, SST receptors being expressed by tumor cells of various tumor histotypes, their pharmacological use is potentially extendible to other cancer types, although to date no significant results have been obtained. In this paper the most recent findings on the expression and functional roles of SST and SST receptors in tumor cells are discussed.

NDM29, a RNA polymerase III-dependent non coding RNA, promotes amyloidogenic processing of APP and amyloid β secretion.

Neuroblastoma Differentiation Marker 29 (NDM29) is a RNA polymerase (pol) III-transcribed non-coding (nc) RNA whose synthesis drives neuroblastoma (NB) cell differentiation to a nonmalignant neuron-like phenotype. Since in this process a complex pattern of molecular changes is associated to plasma membrane protein repertoire we hypothesized that the expression of NDM29 might influence also key players of neurodegenerative pathways. In this work we show that the NDM29-dependent cell maturation induces amyloid precursor protein (APP) synthesis, leading to the increase of amyloid β peptide (Aβ) secretion and the concomitant increment of Aβ x-42/Aβ x-40 ratio. We also demonstrate that the expression of NDM29 RNA, and the consequent increase of Aβ formation, can be promoted by inflammatory stimuli (and repressed by anti-inflammatory drugs). Moreover, NDM29 expression was detected in normal human brains although an abnormal increased synthesis of this ncRNA is induced in patients affected by neurodegenerative diseases. Therefore, the complex of events triggered by NDM29 expression induces a condition that favors the formation of Aβ peptides in the extracellular space, as it may occur in Alzheimers Disease (AD). In addition, these data unexpectedly show that a pol III-dependent small RNA can act as key regulator of brain physiology and/or pathology suggesting that a better knowledge of this portion of the human transcriptome might provide hints for neurodegeneration studies.

Neurodegeneration in Alzheimer Disease: Role of Amyloid Precursor Protein and Presenilin 1 Intracellular Signaling

Alzheimer disease (AD) is a heterogeneous neurodegenerative disorder characterized by (1) progressive loss of synapses and neurons, (2) intracellular neurofibrillary tangles, composed of hyperphosphorylated Tau protein, and (3) amyloid plaques. Genetically, AD is linked to mutations in few proteins amyloid precursor protein (APP) and presenilin 1 and 2 (PS1 and PS2). The molecular mechanisms underlying neurodegeneration in AD as well as the physiological function of APP are not yet known. A recent theory has proposed that APP and PS1 modulate intracellular signals to induce cell-cycle abnormalities responsible for neuronal death and possibly amyloid deposition. This hypothesis is supported by the presence of a complex network of proteins, clearly involved in the regulation of signal transduction mechanisms that interact with both APP and PS1. In this review we discuss the significance of novel finding related to cell-signaling events modulated by APP and PS1 in the development of neurodegeneration.

Characterization of the Proapoptotic Intracellular Mechanisms Induced by a Toxic Conformer of the Recombinant Human Prion Protein Fragment 90–231

Abstract:  Prion diseases comprise a group of fatal neurodegenerative disorders that affect both animals and humans. The transition of the prion protein (PrP) from a mainly α‐structured isoform (PrPC) to a prevalent β‐sheet‐containing protein (PrPSc) is believed to represent a major pathogenetic mechanism in prion diseases. To investigate the linkage between PrP neurotoxicity and its conformation, we used a recombinant prion protein fragment corresponding to the amino acidic sequence 90–231 of human prion protein (hPrP90–231). Using thermal denaturation, we set up an experimental model to induce the process of conversion from PrPC to PrPSc. We report that partial thermal denaturation converts hPrP90–231 into a β‐sheet‐rich isoform, displaying a temperature‐ and time‐dependent conversion into oligomeric structures that share some physico‐chemical characteristics with brain PrPSc. SH‐SY5Y cells were chosen to characterize the potential neurotoxic effect of hPrP90–231 in its different structural conformations. We demonstrated that hPrP90–231 in β‐conformation, but not when α‐structured, powerfully affected the survival of these cells. hPrP90–231 β‐structured caused DNA fragmentation and a significant increase in caspase‐3 proteolytic activity (maximal effects + 170%), suggesting the occurrence of apoptotic cell death. Finally, we investigated the involvement of MAP kinases in the regulation of β‐hPrP90–231‐dependent apoptosis. We observed that the p38 MAP kinase blocker SB203580 prevented the apoptotic cell death evoked by hPrP90–231, and Western blot analysis revealed that the exposure of the cells to the peptide induced p38 phosphorylation. In conclusion, we demonstrate that the hPrP90–231 elicits proapoptotic activity when in β‐sheet‐rich conformation and that this effect is mediated by p38 and caspase‐3 activation.

Cellular prion protein controls stem cell-like properties of human glioblastoma tumor-initiating cells

Prion protein (PrPC) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrPC is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrPC regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrPC in GBM cell pathogenicity focusing on tumor-initiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrPC expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrPC expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrPC down-regulated cells. Moreover, PrPC down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways (i.e. increased GFAP expression). Our results suggest that PrPC controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype.

Role of Prion Protein Aggregation in Neurotoxicity

In several neurodegenerative diseases, such as Parkinson, Alzheimer’s, Huntington, and prion diseases, the deposition of aggregated misfolded proteins is believed to be responsible for the neurotoxicity that characterizes these diseases. Prion protein (PrP), the protein responsible of prion diseases, has been deeply studied for the peculiar feature of its misfolded oligomers that are able to propagate within affected brains, inducing the conversion of the natively folded PrP into the pathological conformation. In this review, we summarize the available experimental evidence concerning the relationship between aggregation status of misfolded PrP and neuronal death in the course of prion diseases. In particular, we describe the main findings resulting from the use of different synthetic (mainly PrP106-126) and recombinant PrP-derived peptides, as far as mechanisms of aggregation and amyloid formation, and how these different spatial conformations can affect neuronal death. In particular, most data support the involvement of non-fibrillar oligomers rather than actual amyloid fibers as the determinant of neuronal death.

Apoptotic cell death influences the signaling activity of the amyloid precursor protein through ShcA and Grb2 adaptor proteins in neuroblastoma SH‐SY5Y cells

The amyloid precursor protein (APP) is an ubiquitous receptor‐like molecule involved in the pathogenesis of Alzheimers disease (AD). APP and some of its C‐terminal proteolytic fragments (CTFs) have been shown to be phosphorylated and to interact with cytosolic phosphotyrosine binding (PTB) domain containing proteins involved in cell signaling and vesicular transport. Among others, the interaction between tyrosine‐phosphorylated CTFs and ShcA‐Grb2 adaptors is highly enhanced in AD brain. Here we have identified in SH‐SY5Y neuroblastoma cells an interaction between APP holoprotein and the adaptor Grb2. Upon activation of apoptotic cell death this interaction is rapidly degraded, APP is partially cleaved and the complex APP/Grb2 is replaced by a new complex between CTFs and ShcA that still involves Grb2. The formation of these complexes is regulated by beta‐site APP‐cleaving enzyme 1 and influences the phosphorylation of mitogen‐activated protein kinase p44/42 extracellular signal‐regulated kinase as well as the level of apoptotic death of the cells. These data suggest a dual role in cell signaling for APP and its CTFs in neuroblastoma cells, in a manner similar to that previously reported for other tyrosine kinase receptor, through a tightly regulated coupling with alternative intracellular adaptors to control the signaling of the cell.

Amyloid Precursor Protein and Presenilin Involvement in Cell Signaling

To date the most relevant role for the amyloid precursor protein (APP) and for the presenilins (PSs) on Alzheimer’s disease (AD) genesis is linked to the ‘amyloid hypothesis’, which considers an aberrant formation of amyloid-β peptides the cause of neurodegeneration. In this view, APP is merely a substrate, cleaved by the γ-secretase complex to form toxic amyloid peptides, PSs are key players in γ-secretase complex, and corollary or secondary events are Tau-linked pathology and gliosis. A second theory, complementary to the amyloid hypothesis, proposes that APP and PSs may modulate a yet unclear cell signal, the disruption of which may induce cell-cycle abnormalities, neuronal death, eventually amyloid formation and finally dementia. This hypothesis is supported by the presence of a complex network of proteins, with a clear relevance for signal transduction mechanisms, which interact with APP or PSs. In this scenario, the C-terminal domain of APP has a pivotal role due to the presence of the 682YENPTY687 motif that represents the docking site for multiple interacting proteins involved in cell signaling. In this review we discuss the significance of novel findings related to cell signaling events modulated by APP and PSs for AD development.