Martín Roffé

Prion protein interaction with stress-inducible protein 1 enhances neuronal protein synthesis via mTOR

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases caused by the conversion of prion protein (PrPC) into an infectious isoform (PrPSc). How this event leads to pathology is not fully understood. Here we demonstrate that protein synthesis in neurons is enhanced via PrPC interaction with stress-inducible protein 1 (STI1). We also show that neuroprotection and neuritogenesis mediated by PrPC–STI1 engagement are dependent upon the increased protein synthesis mediated by PI3K-mTOR signaling. Strikingly, the translational stimulation mediated by PrPC–STI1 binding is corrupted in neuronal cell lines persistently infected with PrPSc, as well as in primary cultured hippocampal neurons acutely exposed to PrPSc. Consistent with this, high levels of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation were found in PrPSc-infected cells and in neurons acutely exposed to PrPSc. These data indicate that modulation of protein synthesis is critical for PrPC–STI1 neurotrophic functions, and point to the impairment of this process during PrPSc infection as a possible contributor to neurodegeneration.

Metabotropic glutamate receptors transduce signals for neurite outgrowth after binding of the prion protein to laminin γ1 chain

The prion protein (PrPC) is highly expressed in the nervous system, and its abnormal con‐former is associated with prion diseases. PrPC is anchored to cell membranes by glycosylphosphatidylinositol, and transmembrane proteins are likely required for PrPC‐mediated intracellular signaling. Binding of laminin (Ln) to PrPC modulates neuronal plasticity and memory. We addressed signaling pathways triggered by PrPC‐Ln interaction in order to identify transmembrane proteins involved in the transduction of PrPC‐Ln signals. The Ln γl‐chain peptide, which contains the Ln binding site for PrPC, induced neuritogenesis through activation of phos‐pholipase C (PLC), Ca2+ mobilization from intracellular stores, and protein kinase C and extracellular signalregulated kinase (ERK1/2) activation in primary cultures of neurons from wild‐type, but not PrPC‐null mice. Phage display, coimmunoprecipitation, and colocalization experiments showed that group I metabotropic glutamate receptors (mGluRl/5) associate with PrPC. Expression of either mGluRl or mGluR5 in HEK293 cells reconstituted the signaling pathways mediated by PrPC‐Ln γl peptide interaction. Specific inhibitors of these receptors impaired PrPC‐Ln γl peptide‐induced signaling and neuri‐togenesis. These data show that group I mGluRs are involved in the transduction of cellular signals triggered by PrPC‐Ln, and they support the notion that PrPC participates in the assembly of multiprotein complexes with physiological functions on neurons.—Beraldo, F. H., Arantes, C. P., Santos, T. G., Machado, C. F., Roffe, M., Hajj, G. N., Lee, K. S., Magalhães, A. C., Caetano, F. A., Mancini, G. L., Lopes, M. H., Amãrico, T. A., Magdesian, M. H., Ferguson, S. S. G., Linden, R., Prado, M. A. M., Martins, V. R. Metabotropic glutamate receptors transduce signals for neurite outgrowth after binding of the prion protein to laminin γl chain. FASEB J. 25, 265–279 (20ll). www.fasebj.org

Stress-inducible phosphoprotein 1 has unique cochaperone activity during development and regulates cellular response to ischemia via the prion protein

Stress‐inducible phosphoprotein 1 (STI1) is part of the chaperone machinery, but it also functions as an extracellular ligand for the prion protein. However, the physiological relevance of these STI1 activities in vivo is unknown. Here, we show that in the absence of embryonic STI1, several Hsp90 client proteins are decreased by 50%, although Hsp90 levels are unaffected. Mutant STI1 mice showed increased caspase‐3 activation and 50% impairment in cellular proliferation. Moreover, placental disruption and lack of cellular viability were linked to embryonic death by E10.5 in STI1‐mutant mice. Rescue of embryonic lethality in these mutants, by transgenic expression of the STI1 gene, supported a unique role for STI1 during embryonic development. The response of STI1 haploinsufficient mice to cellular stress seemed compromised, and mutant mice showed increased vulnerability to ischemic insult. At the cellular level, ischemia increased the secretion of STI1 from wild‐type astrocytes by 3‐fold, whereas STI1 haploinsufficient mice secreted half as much STI1. Interesting, extracellular STI1 prevented ischemia‐mediated neuronal death in a prion protein‐dependent way. Our study reveals essential roles for intracellular and extracellular STI1 in cellular resilience.—Beraldo, F. H., Soares, I. N., Goncalves, D. F., Fan, J., Thomas, A. A., Santos, T. G., Mohammad, A. H., Roffe, M., Calder, M. D., Nikolova, S., Hajj, G. N., Guimaraes, A. N., Massensini, A. R., Welch, I., Betts, D. H., Gros, R., Drangova, M., Watson, A. J., Bartha, R., Prado, V. F., Martins, V. R., and Prado, M. A. M., Stress‐inducible phosphoprotein 1 has unique cochaperone activity during development and regulates cellular response to ischemia via the prion protein. FASEB J. 27, 3594–3607 (2013). www.fasebj.org

IMPACT Is a Developmentally Regulated Protein in Neurons That Opposes the Eukaryotic Initiation Factor 2α Kinase GCN2 in the modulation of Neurite Outgrowth

Background: IMPACT inhibits GCN2, a kinase that directs stress remedial responses by attenuating translation and controls feeding behavior and memory. Results: Neuronal IMPACT is developmentally up-regulated, promoting protein synthesis and neuritogenesis, opposing GCN2. Conclusion: GCN2 and IMPACT modulate an early step in neuronal differentiation. Significance: A neuron-specific developmental program is controlled by two evolutionarily conserved translational regulators. The product of the mouse Imprinted and Ancient gene, IMPACT, is preferentially expressed in neurons. We have previously shown that IMPACT overexpression inhibits the activation of the protein kinase GCN2, which signals amino acid starvation. GCN2 phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in inhibition of general protein synthesis but increased translation of specific messages, such as ATF4. GCN2 is also involved in the regulation of neuronal functions, controlling synaptic plasticity, memory, and feeding behavior. We show here that IMPACT abundance increases during differentiation of neurons and neuron-like N2a cells, whereas GCN2 displays lowered activation levels. Upon differentiation, IMPACT associates with translating ribosomes, enhances translation initiation, and down-regulates the expression of ATF4. We further show that endogenous IMPACT promotes neurite outgrowth whereas GCN2 is a strong inhibitor of spontaneous neuritogenesis. Together, these results uncover the participation of the GCN2-IMPACT module of translational regulation in a highly controlled step in the development of the nervous system.

Laminin-γ1 chain and stress inducible protein 1 synergistically mediate PrPC-dependent axonal growth via Ca2+ mobilization in dorsal root ganglia neurons

Prion protein (PrPC) is a cell surface glycoprotein that is abundantly expressed in nervous system. The elucidation of the PrPC interactome network and its significance on neural physiology is crucial to understanding neurodegenerative events associated with prion and Alzheimers diseases. PrPC co‐opts stress inducible protein 1/alpha7 nicotinic acetylcholine receptor (STI1/α7nAChR) or laminin/Type I metabotropic glutamate receptors (mGluR1/5) to modulate hippocampal neuronal survival and differentiation. However, potential cross‐talk between these protein complexes and their role in peripheral neurons has never been addressed. To explore this issue, we investigated PrPC‐mediated axonogenesis in peripheral neurons in response to STI1 and laminin‐γ1 chain‐derived peptide (Ln‐γ1). STI1 and Ln‐γ1 promoted robust axonogenesis in wild‐type neurons, whereas no effect was observed in neurons from PrPC‐null mice. PrPC binding to Ln‐γ1 or STI1 led to an increase in intracellular Ca2+ levels via distinct mechanisms: STI1 promoted extracellular Ca2+ influx, and Ln‐γ1 released calcium from intracellular stores. Both effects depend on phospholipase C activation, which is modulated by mGluR1/5 for Ln‐γ1, but depends on, C‐type transient receptor potential (TRPC) channels rather than α7nAChR for STI1. Treatment of neurons with suboptimal concentrations of both ligands led to synergistic actions on PrPC‐mediated calcium response and axonogenesis. This effect was likely mediated by simultaneous binding of the two ligands to PrPC. These results suggest a role for PrPC as an organizer of diverse multiprotein complexes, triggering specific signaling pathways and promoting axonogenesis in the peripheral nervous system.

GCN2 activation and eIF2α phosphorylation in the maturation of mouse oocytes

GCN2 is one of the four mammalian kinases that phosphorylate the alpha subunit of the translation initiation factor 2 (eIF2alpha) in a variety of stress situations, resulting in protein synthesis inhibition. GCN2 is involved in regulating metabolism, feeding behavior and memory in rodents. We show here that, relative to other cells, the beta isoform of the GCN2 transcript and the GCN2 protein are highly abundant in unfertilized mouse eggs. In addition, GCN2 in these cells is active, resulting in elevated levels of phosphorylated eIF2alpha. After fertilization, eIF2alpha phosphorylation decreases drastically. These results suggest that GCN2 mediated translational control may contribute to regulatory mechanisms operating during oocyte maturation.

Evaluation of Akt and RICTOR Expression Levels in Astrocytomas of All Grades.

The mammalian target of rapamycin (mTOR) binds to several protein partners and forms two complexes, termed mTOR complexes 1 and 2 (mTORC1/2), that differ in components, substrates, and regulation. mTORC2 contains the protein Rapamycin-insensitive companion of mTOR (RICTOR); phosphorylates kinases of the AGC family, such as Akt; and controls the cytoskeleton. Even though the regulation of mTORC2 activity remains poorly understood, the hyperactivation of this signaling pathway has been shown to contribute to the oncogenic properties of gliomas in experimental models. In this work, we evaluated expression and phosphorylation of Akt, and expression of RICTOR and Ki-67 in 195 human astrocytomas of different grades (38 cases of grade I, 49 grade II, 15 grade III, and 93 grade IV) and 30 normal brains. Expression and phosphorylation of Akt increased with histological grade and correlated with a worse overall survival in glioblastomas (GBMs). RICTOR was overexpressed in grade I and II astrocytomas and demonstrated a shift to nuclear localization in GBMs. Nuclear RICTOR was associated to increased proliferation in GBMs. Our results point to an increase in total and phosphorylated Akt in high-grade gliomas and to a possible role of RICTOR in proliferations of high-grade GBM cells.

Disease-associated Mutations in the Prion Protein Impair Laminin-induced Process Outgrowth and Survival

Background: In addition to the toxicity mediated by prion protein misfolding, mechanisms associated with its loss-of-function in genetic prion diseases are unknown. Results: Neural cells expressing PrPC mutants associated with prion diseases present impaired laminin-mediated process outgrowth and survival. Conclusion: PrPC mutants show loss-of-function in neural cells. Significance: The impairment of prion protein functions may contribute to the etiology of prion diseases. Prions, the agents of transmissible spongiform encephalopathies, require the expression of prion protein (PrPC) to propagate disease. PrPC is converted into an abnormal insoluble form, PrPSc, that gains neurotoxic activity. Conversely, clinical manifestations of prion disease may occur either before or in the absence of PrPSc deposits, but the loss of normal PrPC function contribution for the etiology of these diseases is still debatable. Prion disease-associated mutations in PrPC represent one of the best models to understand the impact of PrPC loss-of-function. PrPC associates with various molecules and, in particular, the interaction of PrPC with laminin (Ln) modulates neuronal plasticity and memory formation. To assess the functional alterations associated with PrPC mutations, wild-type and mutated PrPC proteins were expressed in a neural cell line derived from a PrPC-null mouse. Treatment with the laminin γ1 chain peptide (Ln γ1), which mimics the Ln binding site for PrPC, increased intracellular calcium in cells expressing wild-type PrPC, whereas a significantly lower response was observed in cells expressing mutated PrPC molecules. The Ln γ1 did not promote process outgrowth or protect against staurosporine-induced cell death in cells expressing mutated PrPC molecules in contrast to cells expressing wild-type PrPC. The co-expression of wild-type PrPC with mutated PrPC molecules was able to rescue the Ln protective effects, indicating the lack of negative dominance of PrPC mutated molecules. These results indicate that PrPC mutations impair process outgrowth and survival mediated by Ln γ1 peptide in neural cells, which may contribute to the pathogenesis of genetic prion diseases.

PHF21B as a candidate tumor suppressor gene in head and neck squamous cell carcinomas

A significant association between DNA losses on 22q13.31 and head and neck squamous cell carcinomas (HNSCC) was previously reported by our group. Our data indicated that PHF21B gene, mapped on 22q13.31 and encoding a protein with function of chromatin‐mediated transcriptional regulation, might be a putative tumor suppressor gene. To test this hypothesis, gene copy number was assessed in 75 HNSCC and 49 matched peripheral blood samples. PHF21B losses were detected in 43 tumors and were significantly associated with patients with familial history of cancer (P < 0.0001); i.e., 36/43 cases showed a positive family history of cancer and 22/36 had first‐degree relatives with cancer (P = 0.049). In attempt to investigate other mechanisms for PHF21B loss of function, DNA sequencing was performed and no mutations were detected. We next evaluated the gene expression levels after inhibition of DNA methylation in nine HNSCC and breast carcinoma cell lines. Additionally, PHF21B expression levels were evaluated in colon cancer HCT116 cells as well as in its counterpart DKO (double knockout of DNMT1 and DNMT3B). The higher expression levels of PHF21B gene detected in DKO cells were inversely correlated with the DNA methylation. Further, DNA methylation in the specific promoter‐associated CpG Island was investigated. Interestingly, gene hypermethylation was detected in 13/37 tumors: 5/13 HNSCC cases had family history of cancer in first‐degree relatives and 8/13 showed both, DNA methylation and PHF21B losses in the tumor sample. One patient had PHF21B loss in the peripheral blood cells and PHF21B methylation in the tumor sample. Additionally, overexpression of PHF21B in cell lines drastically reduces clonogenic and migratory abilities. These data suggest that PHF21B is a novel tumor suppressor gene that can be inactivated by genetic and epigenetic mechanisms in the human cancer.

Polysome-profiling in small tissue samples

Abstract Polysome-profiling is commonly used to study translatomes and applies laborious extraction of efficiently translated mRNA (associated with >3 ribosomes) from a large volume across many fractions. This property makes polysome-profiling inconvenient for larger experimental designs or samples with low RNA amounts. To address this, we optimized a non-linear sucrose gradient which reproducibly enriches for efficiently translated mRNA in only one or two fractions, thereby reducing sample handling 5–10-fold. The technique generates polysome-associated RNA with a quality reflecting the starting material and, when coupled with smart-seq2 single-cell RNA sequencing, translatomes in small tissues from biobanks can be obtained. Translatomes acquired using optimized non-linear gradients resemble those obtained with the standard approach employing linear gradients. Polysome-profiling using optimized non-linear gradients in serum starved HCT-116 cells with or without p53 showed that p53 status associates with changes in mRNA abundance and translational efficiency leading to changes in protein levels. Moreover, p53 status also induced translational buffering whereby changes in mRNA levels are buffered at the level of mRNA translation. Thus, here we present a polysome-profiling technique applicable to large study designs, primary cells and frozen tissue samples such as those collected in biobanks.