Alliance Against Cancer, the network of Italian cancer centers bridging research and care.

Alliance Against Cancer (ACC) was established in Rome in 2002 as a consortium of six Italian comprehensive cancer centers (Founders). The aims of ACC were to promote a network among Italian oncologic institutions in order to develop specific, advanced projects in clinical and translational research. During the following years, many additional full and associate members joined ACC, that presently includes the National Institute of Health, 17 research-oriented hospitals, scientific and patient organizations. Furthermore, in the last three years ACC underwent a reorganization process that redesigned the structure, governance and major activities. The present goal of ACC is to achieve high standards of care across Italy, to implement and harmonize principles of modern personalized and precision medicine, by developing cost effective processes and to provide tailored information to cancer patients. We herein summarize some of the major initiatives that ACC is currently developing to reach its goal, including tumor genetic screening programs, establishment of clinical trial programs for cancer patients treated in Italian cancer centers, facilitate their access to innovative drugs under development, improve quality through an European accreditation process (European Organization of Cancer Institutes), and develop international partnerships. In conclusion, ACC is a growing organization, trying to respond to the need of networking in Italy and may contribute significantly to improve the way we face cancer in Europe.

The DNA demethylating agent decitabine activates the TRAIL pathway and induces apoptosis in acute myeloid leukemia.

Although epigenetic drugs have been approved for use in selected malignancies, there is significant need for a better understanding of their mechanism of action. Here, we study the action of a clinically approved DNA-methyltransferase inhibitor - decitabine (DAC) - in acute myeloid leukemia (AML) cells. At low doses, DAC treatment induced apoptosis of NB4 Acute Promyelocytic Leukemia (APL) cells, which was associated with the activation of the extrinsic apoptotic pathway. Expression studies of the members of the Death Receptor family demonstrated that DAC induces the expression of TNF-related apoptosis-inducing ligand (TRAIL). Upregulation of TRAIL, upon DAC treatment, was associated with specific epigenetic modifications induced by DAC in the proximity of the TRAIL promoter, as demonstrated by DNA demethylation, increased DNaseI sensitivity and histone acetylation of a non-CpG island, CpG-rich region located 2kb upstream to the transcription start site. Luciferase assay experiments showed that this region behave as a DNA methylation sensitive transcriptional regulatory element. The CpG regulatory element was also found methylated in samples derived from APL patients. These findings have been confirmed in the non-APL, AML Kasumi cell line, suggesting that this regulatory mechanism may be extended to other AMLs. Our study suggests that DNA methylation is a regulatory mechanism relevant for silencing of the TRAIL apoptotic pathway in leukemic cells, and further elucidates the mechanism by which epigenetic drugs mediate their anti-leukemic effects.

S1P1 expression is controlled by the pro-oxidant activity of p66Shc and is impaired in B-CLL patients with unfavorable prognosis.

Although intrinsic apoptosis defects are causal to the extended survival of chronic lymphocytic leukemia (CLL) B cells, several lines of evidence support a contribution of the peripheral lymphoid organs and BM microenvironment to the extended lifespan of leukemic B cells. Lymphocyte trafficking is controlled by homing signals provided by stromal cell-derived chemokines and egress signals provided by sphingosine-1-phosphate (S1P). In the present study, we show that expression of S1P1, the S1P receptor responsible for lymphocyte egress, is selectively reduced in CLL B cells with unmutated IGHV. Expression of S1P2, which controls B-cell homeostasis, is also impaired in CLL B cells but independently of the IGHV mutational status. We provide evidence herein that p66Shc, a Shc adaptor family member the deficiency of which is implicated in the apoptosis defects of CLL B cells, controls S1P1 expression through its pro-oxidant activity. p66Shc also controls the expression of the homing receptor CCR7, which opposes S1P1 by promoting lymphocyte retention in peripheral lymphoid organs. The results of the present study provide insights into the regulation of S1P1 expression in B cells and suggest that defective egress caused by impaired S1P1 expression contributes to the extended survival of CLL B cells by prolonging their residency in the prosurvival niche of peripheral lymphoid organs.

Time makes histone H3 modifications drift in mouse liver.

To detect the epigenetic drift of time passing, we determined the genome-wide distributions of mono- and tri-methylated lysine 4 and acetylated and tri-methylated lysine 27 of histone H3 in the livers of healthy 3, 6 and 12 months old C57BL/6 mice. The comparison of different age profiles of histone H3 marks revealed global redistribution of histone H3 modifications with time, in particular in intergenic regions and near transcription start sites, as well as altered correlation between the profiles of different histone modifications. Moreover, feeding mice with caloric restriction diet, a treatment known to retard aging, reduced the extent of changes occurring during the first year of life in these genomic regions.

Cyclophosphamide and Vinorelbine Activate Stem-Like CD8+ T Cells and Improve Anti-PD-1 Efficacy in Triple-Negative Breast Cancer.

Checkpoint inhibitors (CI) instigate anticancer immunity in many neoplastic diseases, albeit only in a fraction of patients. The clinical success of cyclophosphamide (C)-based haploidentical stem-cell transplants indicates that this drug may re-orchestrate the immune system. Using models of triple-negative breast cancer (TNBC) with different intratumoral immune contexture, we demonstrate that a combinatorial therapy of intermittent C, CI, and vinorelbine activates antigen-presenting cells (APC), and abrogates local and metastatic tumor growth by a T-cell-related effect. Single-cell transcriptome analysis of >50,000 intratumoral immune cells after therapy treatment showed a gene signature suggestive of a change resulting from exposure to a mitogen, ligand, or antigen for which it is specific, as well as APC-to-T-cell adhesion. This transcriptional program also increased intratumoral Tcf1+ stem-like CD8+ T cells and altered the balance between terminally and progenitor-exhausted T cells favoring the latter. Overall, our data support the clinical investigation of this therapy in TNBC. SIGNIFICANCE: A combinatorial therapy in mouse models of breast cancer increases checkpoint inhibition by activating antigen-presenting cells, enhancing intratumoral Tcf1+ stem-like CD8+ T cells, and increasing progenitor exhausted CD8+ T cells.

The cardioprotective effects elicited by p66(Shc) ablation demonstrate the crucial role of mitochondrial ROS formation in ischemia/reperfusion injury.

Although a major contribution to myocardial ischemia-reperfusion (I/R) injury is suggested to be provided by formation of reactive oxygen species (ROS) within mitochondria, sites and mechanisms are far from being elucidated. Besides a dysfunctional respiratory chain, other mitochondrial components, such as monoamine oxidase and p66(Shc), might be involved in oxidative stress. In particular, p66(Shc) has been shown to catalyze the formation of H(2)O(2). The relationship among p66(Shc), ROS production and cardiac damage was investigated by comparing hearts from p66(Shc) knockout mice (p66(Shc-/-)) and wild-type (WT) littermates. Perfused hearts were subjected to 40 min of global ischemia followed by 15 min of reperfusion. Hearts devoid of p66(Shc) were significantly protected from I/R insult as shown by (i) reduced release of lactate dehydrogenase in the coronary effluent (25.7+/-7.49% in p66(Shc-/-) vs. 39.58+/-5.17% in WT); (ii) decreased oxidative stress as shown by a 63% decrease in malondialdehyde formation and 40+/-8% decrease in tropomyosin oxidation. The degree of protection was independent of aging. The cardioprotective efficacy associated with p66(Shc) ablation was comparable with that afforded by other antioxidant interventions and could not be increased by antioxidant co-administration suggesting that p66(Shc) is downstream of other pathways involved in ROS formation. In addition, the absence of p66(Shc) did not affect the protection afforded by ischemic preconditioning. In conclusion, the absence of p66(Shc) reduces the susceptibility to reperfusion injury by preventing oxidative stress. The present findings provide solid and direct evidence that mitochondrial ROS formation catalyzed by p66(Shc) is causally related to reperfusion damage.

Master protocols in immuno-oncology: do novel drugs deserve novel designs?

The rapid rise to fame of immuno-oncology (IO) drugs has generated unprecedented interest in the industry, patients and doctors, and has had a major impact in the treatment of most cancers. An interesting aspect in the clinical development of many IO agents is the increasing reliance on nonconventional trial design, including the so-called 'master protocols' that incorporate various adaptive features and often heavily rely on biomarkers to select patient populations most likely to benefit. These novel designs promise to maximize the clinical benefit that can be reaped from clinical research, but are not without costs. Their acceptance as solid evidence basis for use outside of the research context requires profound cultural changes by multiple stakeholders, including regulatory bodies, decision-makers, statisticians, researchers, doctors and, most importantly, patients. Here we review characteristics of recent and ongoing trials testing IO drugs with unconventional design, and we highlight trends and critical aspects.

RaLP, a new member of the Src homology and collagen family, regulates cell migration and tumor growth of metastatic melanomas.

The Src homology and collagen (Src) family of adaptor proteins comprises six Shc-like proteins encoded by three loci in mammals (Shc, Rai, and Sli). Shc-like proteins are tyrosine kinase substrates, which regulate diverse signaling pathways and cellular functions, including Ras and proliferation (p52/p46Shc), phosphatidylinositol 3-kinase and survival (p54Rai), and mitochondrial permeability transition and apoptosis (p66Shc). Here, we report the identification, cloning, and sequence characterization of a new member of the Shc family that we termed RaLP. RaLP encodes a 69-kDa protein characterized by the CH2-PTB-CH1-SH2 modularity, typical of the Shc protein family, and expressed, among adult tissues, only in melanomas. Analysis of RaLP expression during the melanoma progression revealed low expression in normal melanocytes and benign nevi, whereas high levels of RaLP protein were found at the transition from radial growth phase to vertical growth phase and metastatic melanomas, when tumor cells acquire migratory competence and invasive potential. Notably, silencing of RaLP expression in metastatic melanomas by RNA interference reduced tumorigenesis in vivo. Analysis of RaLP in melanoma signal transduction pathways revealed that (a) when ectopically expressed in RaLP-negative melanocytes and nonmetastatic melanoma cells, it functions as a substrate of activated insulin-like growth factor-1 and epidermal growth factor receptors and increases Ras/mitogen-activated protein kinase (MAPK) signaling and cell migration, whereas (b) its silencing in RaLP-positive melanoma cells abrogates cell migration in vitro, without affecting MAPK signaling, suggesting that RaLP activates both Ras-dependent and Ras-independent migratory pathways in melanomas. These findings indicate that RaLP is a specific marker of metastatic melanomas, a critical determinant in the acquisition of the migratory phenotype by melanoma cells, and a potential target for novel anti-melanoma therapeutic strategies.

Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.

BACKGROUND: Nucleophosmin (NPM), a nucleocytoplasmic shuttling protein with prominent nucleolar localization, regulates the ARF-p53 tumor-suppressor pathway. Translocations involving the NPM gene cause cytoplasmic dislocation of the NPM protein. METHODS: We used immunohistochemical methods to study the subcellular localization of NPM in bone marrow-biopsy specimens from 591 patients with primary acute myelogenous leukemia (AML). We then correlated the presence of cytoplasmic NPM with clinical and biologic features of the disease. RESULTS: Cytoplasmic NPM was detected in 208 (35.2 percent) of the 591 specimens from patients with primary AML but not in 135 secondary AML specimens or in 980 hematopoietic or extrahematopoietic neoplasms other than AML. It was associated with a wide spectrum of morphologic subtypes of the disease, a normal karyotype, and responsiveness to induction chemotherapy, but not with recurrent genetic abnormalities. There was a high frequency of FLT3 internal tandem duplications and absence of CD34 and CD133 in AML specimens with a normal karyotype and cytoplasmic dislocation of NPM, but not in those in which the protein was restricted to the nucleus. AML specimens with cytoplasmic NPM carried mutations of the NPM gene that were predicted to alter the protein at its C-terminal; this mutant gene caused cytoplasmic localization of NPM in transfected cells. CONCLUSIONS: Cytoplasmic NPM is a characteristic feature of a large subgroup of patients with AML who have a normal karyotype, NPM gene mutations, and responsiveness to induction chemotherapy.

Precision Trial Drawer, a Computational Tool to Assist Planning of Genomics-Driven Trials in Oncology.

PURPOSE: Trials that accrue participants on the basis of genetic biomarkers are a powerful means of testing targeted drugs, but they are often complicated by the rarity of the biomarker-positive population. Umbrella trials circumvent this by testing multiple hypotheses to maximize accrual. However, bigger trials have higher chances of conflicting treatment allocations because of the coexistence of multiple actionable alterations; allocation strategies greatly affect the efficiency of enrollment and should be carefully planned on the basis of relative mutation frequencies, leveraging information from large sequencing projects. METHODS: We developed software named Precision Trial Drawer (PTD) to estimate parameters that are useful for designing precision trials, most importantly, the number of patients needed to molecularly screen (NNMS) and the allocation rule that maximizes patient accrual on the basis of mutation frequency, systematically assigning patients with conflicting allocations to the drug associated with the rarer mutation. We used data from The Cancer Genome Atlas to show their potential in a 10-arm imaginary trial of multiple cancers on the basis of genetic alterations suggested by the past Molecular Analysis for Personalised Therapy (MAP) conference. We validated PTD predictions versus real data from the SHIVA (A Randomized Phase II Trial Comparing Therapy Based on Tumor Molecular Profiling Versus Conventional Therapy in Patients With Refractory Cancer) trial. RESULTS: In the MAP imaginary trial, PTD-optimized allocation reduces number of patients needed to molecularly screen by up to 71.8% (3.5 times) compared with nonoptimal trial designs. In the SHIVA trial, PTD correctly predicted the fraction of patients with actionable alterations (33.51% [95% CI, 29.4% to 37.6%] in imaginary v 32.92% [95% CI, 28.2% to 37.6%] expected) and allocation to specific treatment groups (RAS/MEK, PI3K/mTOR, or both). CONCLUSION: PTD correctly predicts crucial parameters for the design of multiarm genetic biomarker-driven trials. PTD is available as a package in the R programming language and as an open-access Web-based app. It represents a useful resource for the community of precision oncology trialists. The Web-based app is available at https://gmelloni.github.io/ptd/shinyapp.html.

Deletion of p66Shc longevity gene protects against experimental diabetic glomerulopathy by preventing diabetes-induced oxidative stress.

p66(Shc) regulates both steady-state and environmental stress-dependent reactive oxygen species (ROS) generation. Its deletion was shown to confer resistance to oxidative stress and protect mice from aging-associated vascular disease. This study was aimed at verifying the hypothesis that p66(Shc) deletion also protects from diabetic glomerulopathy by reducing oxidative stress. Streptozotocin-induced diabetic p66(Shc) knockout (KO) mice showed less marked changes in renal function and structure, as indicated by the significantly lower levels of proteinuria, albuminuria, glomerular sclerosis index, and glomerular and mesangial areas. Glomerular content of fibronectin and collagen IV was also lower in diabetic KO versus wild-type mice, whereas apoptosis was detected only in diabetic wild-type mice. Serum and renal tissue advanced glycation end products and plasma isoprostane 8-epi-prostaglandin F2alpha levels and activation of nuclear factor kappaB (NF-kappaB) were also lower in diabetic KO than in wild-type mice. Mesangial cells from KO mice grown under high-glucose conditions showed lower cell death rate, matrix production, ROS levels, and activation of NF-kappaB than those from wild-type mice. These data support a role for oxidative stress in the pathogenesis of diabetic glomerulopathy and indicate that p66(Shc) is involved in the molecular mechanism(s) underlying diabetes-induced oxidative stress and oxidant-dependent renal injury.

p66SHC promotes apoptosis and antagonizes mitogenic signaling in T cells.

Of the three Shc isoforms, p66Shc is responsible for fine-tuning p52/p46Shc signaling to Ras and has been implicated in apoptotic responses to oxidative stress. Here we show that human peripheral blood lymphocytes and mouse thymocytes and splenic T cells acquire the capacity to express p66Shc in response to apoptogenic stimulation. Using a panel of T-cell transfectants and p66Shc(-/-) T cells, we show that p66Shc expression results in increased susceptibility to apoptogenic stimuli, which depends on Ser36 phosphorylation and correlates with an altered balance in apoptosis-regulating gene expression. Furthermore, p66Shc blunts mitogenic responses to T-cell receptor engagement, at least in part by transdominant inhibition of p52Shc signaling to Ras/mitogen-activated protein kinases, in an S36-dependent manner. The data highlight a novel interplay between p66Shc and p52Shc in the control of T-cell fate.

NGF controls APP cleavage by downregulating APP phosphorylation at Thr668: relevance for Alzheimer's disease.

NGF has been implicated in forebrain neuroprotection from amyloidogenesis and Alzheimer's disease (AD). However, the underlying molecular mechanisms are still poorly understood. Here, we investigated the role of NGF signalling in the metabolism of amyloid precursor protein (APP) in forebrain neurons using primary cultures of septal neurons and acute septo-hippocampal brain slices. In this study, we show that NGF controls the basal level of APP phosphorylation at Thr668 (T668) by downregulating the activity of the Ser/Thr kinase JNK(p54) through the Tyr kinase signalling adaptor SH2-containing sequence C (ShcC). We also found that the specific NGF receptor, Tyr kinase A (TrkA), which is known to bind to APP, fails to interact with the fraction of APP molecules phosphorylated at T668 (APP(pT668) ). Accordingly, the amount of TrkA bound to APP is significantly reduced in the hippocampus of ShcC KO mice and of patients with AD in which elevated APP(pT668) levels are detected. NGF promotes TrkA binding to APP and APP trafficking to the Golgi, where APP-BACE interaction is hindered, finally resulting in reduced generation of sAPPß, CTFß and amyloid-beta (1-42). These results demonstrate that NGF signalling directly controls basal APP phosphorylation, subcellular localization and BACE cleavage, and pave the way for novel approaches specifically targeting ShcC signalling and/or the APP-TrkA interaction in AD therapy.

In Vivo Genetic Screens of Patient-Derived Tumors Revealed Unexpected Frailty of the Transformed Phenotype.

UNLABELLED: The identification of genes maintaining cancer growth is critical to our understanding of tumorigenesis. We report the first in vivo genetic screen of patient-derived tumors, using metastatic melanomas and targeting 236 chromatin genes by expression of specific shRNA libraries. Our screens revealed unprecedented numerosity of genes indispensable for tumor growth (∼50% of tested genes) and unexpected functional heterogeneity among patients (<15% in common). Notably, these genes were not activated by somatic mutations in the same patients and are therefore distinguished from mutated cancer driver genes. We analyzed underlying molecular mechanisms of one of the identified genes, the Histone-lysine N-methyltransferase KMT2D, and showed that it promotes tumorigenesis by dysregulating a subset of transcriptional enhancers and target genes involved in cell migration. The assembly of enhancer genomic patterns by activated KMT2D was highly patient-specific, regardless of the identity of transcriptional targets, suggesting that KMT2D might be activated by distinct upstream signaling pathways. SIGNIFICANCE: Drug targeting of biologically relevant cancer-associated mutations is considered a critical strategy to control cancer growth. Our functional in vivo genetic screens of patient-derived tumors showed unprecedented numerosity and interpatient heterogeneity of genes that are essential for tumor growth, but not mutated, suggesting that multiple, patient-specific signaling pathways are activated in tumors. Cancer Discov; 6(6); 650-63. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 561.

Cell-line specific chromatin acetylation at the Sox10-Pax3 enhancer site modulates the RET proto-oncogene expression.

The RET gene is expressed with high tissue and stage specificity during development. Understanding its transcriptional regulation might provide new clues to clarify developmental mechanisms. Here we show that the histone deacetylase inhibitor sodium butyrate (NaB) increases RET transcription in cells displaying low levels of its mRNA, while it has no effect in cells expressing at high levels. Chromatin immunoprecipitation (ChIP) experiments showed increased histone acetylation within the 5' flanking [corrected] region, in particular the Sox10-Pax3 enhancer site, due to NaB. Accordingly, ChIP showed different acetylation levels at the Sox10-Pax3 site associated with cell-line specific RET transcription rates. Concluding, chromatin acetylation targeted to functional sequences in the RET regulatory region may control its transcription.

High-resolution cytometry for high-content cell cycle analysis.

One of the major limitations of flow cytometry (FCM) is the absence of an intracellular view. Automated microscopy and image analysis, together with technological developments, led to new approaches in cytometry that bypass the above limitation, introducing high resolution, high content, and large statistical sampling. However, few attempts have been made, until now, to translate the wide repertoire of FCM assays into high-content image screening. This unit describes the implementation of an acquisition and analysis protocol for evaluation of the cell cycle by automated microscopy. The approach grants the possibility to perform simultaneous analysis of a high number of different parameters. A large part of this unit is devoted to the description of hardware features that can optimize the recorded information together with the acquisition and analysis procedures employed to produce good-quality data.

Confocal microscopy for high-resolution and high-content analysis of the cell cycle.

Optical fluorescence microscopy offers a wide range of technological solutions to address many questions in biomedical research. Spatial resolution has been greatly improved by the use of confocal microscopes, providing a 3-D analysis of the intracellular space. Automation has contributed to make confocal analysis available for high-content image cytometry studies. However, the storage, browsing, and analysis of the amount of data generated can challenge the feasibility of such studies. Presented in this chapter is a multistep acquisition and analysis protocol that can bypass such difficulties by an analysis-driven data collection. Cell-cycle analysis of low-resolution data can be employed to select cell populations of interest that can then be imaged at extremely high resolution and subjected to high-content analysis.

Stem cell transplantation effectively occludes bronchopleural fistula in an animal model.

BACKGROUND: Bronchopleural fistula after lung resection still represents a challenging life-threatening complication for thoracic surgeons. Considering its extremely high mortality rate, an effective treatment is urgently required. Our project investigated the hypothesis of experimental bronchopleural fistula closure by bronchoscopic injection of autologous bone marrow-derived mesenchymal stem cells into the cavity of the fistula, evaluating its feasibility and safety in a large animal model. METHODS: An experimental bronchopleural fistula was created in 9 goats after right upper tracheal lobectomy. The animals were randomly assigned to two groups: one received autologous bone marrow-derived mesenchymal stem cell bronchoscopic transplantation; the other received standard bronchoscopic fibrin glue injection. RESULTS: All animals receiving bronchoscopic stem cell transplantation presented fistula closure by extraluminal fibroblast proliferation and collagenous matrix development; none (0%) died during the study period. All animals receiving standard treatment still presented bronchopleural fistula; 2 of them (40%) died. Findings were confirmed by pathology examination, computed tomography, and magnetic resonance imaging. CONCLUSIONS: Bronchoscopic transplantation of bone marrow-derived mesenchymal stem cells effectively closes experimental bronchopleural fistula by extraluminal fibroblast proliferation and collagenous matrix development. Stem cells may play a crucial role in the treatment of postresectional bronchopleural fistula after standard lung resection. Although these results provide a basis for the development of clinical therapeutic strategies, the exact mechanism by which they are obtained is not yet completely clear; further studies are required to understand exactly how stem cells work in this field.

Diabetes causes bone marrow autonomic neuropathy and impairs stem cell mobilization via dysregulated p66Shc and Sirt1.

Diabetes compromises the bone marrow (BM) microenvironment and reduces the number of circulating CD34(+) cells. Diabetic autonomic neuropathy (DAN) may impact the BM, because the sympathetic nervous system is prominently involved in BM stem cell trafficking. We hypothesize that neuropathy of the BM affects stem cell mobilization and vascular recovery after ischemia in patients with diabetes. We report that, in patients, cardiovascular DAN was associated with fewer circulating CD34(+) cells. Experimental diabetes (streptozotocin-induced and ob/ob mice) or chemical sympathectomy in mice resulted in BM autonomic neuropathy, impaired Lin(-)cKit(+)Sca1(+) (LKS) cell and endothelial progenitor cell (EPC; CD34(+)Flk1(+)) mobilization, and vascular recovery after ischemia. DAN increased the expression of the 66-kDa protein from the src homology and collagen homology domain (p66Shc) and reduced the expression of sirtuin 1 (Sirt1) in mice and humans. p66Shc knockout (KO) in diabetic mice prevented DAN in the BM, and rescued defective LKS cell and EPC mobilization. Hematopoietic Sirt1 KO mimicked the diabetic mobilization defect, whereas hematopoietic Sirt1 overexpression in diabetes rescued defective mobilization and vascular repair. Through p66Shc and Sirt1, diabetes and sympathectomy elevated the expression of various adhesion molecules, including CD62L. CD62L KO partially rescued the defective stem/progenitor cell mobilization. In conclusion, autonomic neuropathy in the BM impairs stem cell mobilization in diabetes with dysregulation of the life-span regulators p66Shc and Sirt1.

The P66Shc/mitochondrial permeability transition pore pathway determines neurodegeneration.

Mitochondrial-mediated oxidative stress and apoptosis play a crucial role in neurodegenerative disease and aging. Both mitochondrial permeability transition (PT) and swelling of mitochondria have been involved in neurodegeneration. Indeed, knockout mice for cyclophilin-D (Cyc-D), a key regulatory component of the PT pore (PTP) that triggers mitochondrial swelling, resulted to be protected in preclinical models of multiple sclerosis (MS), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). However, how neuronal stress is transduced into mitochondrial oxidative stress and swelling is unclear. Recently, the aging determinant p66Shc that generates H2O2 reacting with cytochrome c and induces oxidation of PTP and mitochondrial swelling was found to be involved in MS and ALS. To investigate the role of p66Shc/PTP pathway in neurodegeneration, we performed experimental autoimmune encephalomyelitis (EAE) experiments in p66Shc knockout mice (p66Shc-/-), knock out mice for cyclophilin-D (Cyc-D-/-), and p66Shc Cyc-D double knock out (p66Shc/Cyc-D-/-) mice. Results confirm that deletion of p66Shc protects from EAE without affecting immune response, whereas it is not epistatic to the Cyc-D mutation. These findings demonstrate that p66Shc contributes to EAE induced neuronal damage most likely through the opening of PTP suggesting that p66Shc/PTP pathway transduces neurodegenerative stresses.