BUSCADOR RECOLECTA

Adenovirus Delta-24-RGD has shown a remarkable efficacy in a phase I clinical trial for glioblastoma. Delta-24-RGD induces autophagy in glioma cells, however, the molecular derangements associated with Delta-24-RGD infection remains poorly understood. Here, proteomics was applied to characterize the glioma metabolic disturbances soon after Delta-24-RGD internalization and late in infection. Minutes post-infection, a rapid survival reprogramming of glioma cells was evidenced by an early c-Jun activation and a time-dependent dephosphorylation of multiple survival kinases. At 48 h post-infection (hpi), a severe intracellular proteostasis impairment was characterized, detecting differentially expressed proteins related to mRNA splicing, cytoskeletal organization, oxidative response, and inflammation. Specific kinase-regulated protein interactomes for Delta-24-RGD-modulated proteome revealed interferences with the activation dynamics of protein kinases C and A (PKC, PKA), tyrosine-protein kinase Src (c-Src), glycogen synthase kinase-3 (GSK-3) as well as serine/threonine-protein phosphatases 1 and 2A (PP1, PP2A) at 48hpi, in parallel with adenoviral protein overproduction. Moreover, the late activation of the nuclear factor kappa B (NF-κB) correlates with the extracellular increment of specific cytokines involved in migration, and activation of different inflammatory cells. Taken together, our integrative analysis provides further insights into the effects triggered by Delta-24-RGD in the modulation of tumor suppression and immune response against glioma. Significance: The current study provides new insights regarding the molecular mechanisms governing the glioma metabolism during Delta-24-RGD oncolytic adenoviral therapy. The compilation and analysis of intracellular and extracellular proteomics have led us to characterize: i) the cell survival reprogramming during Delta-24-RGD internalization, ii) the proteostatic disarrangement induced by Delta-24-RGD during the autophagic stage, iii) the protein interactomes for Delta-24-RGD-modulated proteome, iv) the regulatory effects on kinase dynamics induced by Delta-24-RGD late in infection, and v) the overproduction of multitasking cytokines upon Delta-24-RGD treatment. We consider that the quantitative molecular maps generated in this study may establish the foundations for the development of complementary adenoviral based-vectors to increase the potency against glioma., This work was funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (Ref. SAF2014-59340-R To ES), the Department of Economic Development from Government of Navarra (Ref. PC023-PC024, PC025, PC081-82 and PI059 to ES and PI031 to JFI), the Instituto de Salud Carlos III and Fondos Feder Europeos (PI16/00066 to MMA), the Spanish Ministry of Science and Innovation (IEDI-2015-00638 to MMA), the Department of Health of the Government of Navarra (to MMA), the Basque Foundation for Health Research (BIOEF, BIO13/CI/005 to MMA), Asociación Pablo Ugarte-Fuerza, Julen (to MMA). AGM was supported by PEJ-2014-A-61949 (MINECO), and a pre-doctoral fellowship from Public University of Navarra (UPNA). The Proteomics Unit of Navarrabiomed is a member of Proteored, PRB3-ISCIII, and is supported by grant PT17/0019/0009, of the PE I+D+I 2013-2016 funded by ISCIII and FEDER.

The olfactory bulb (OB) is the first processing station in the olfactory pathway. Despite smell impairment, which is considered an early event in Alzheimer’s disease (AD), little is known about the initial molecular disturbances that accompany the AD development at olfactory level. We have interrogated the time-dependent OB molecular landscape in Tg2576 AD mice prior to the appearance of neuropathological amyloid plaques (2-, and 6-month-old), using combinatorial omics analysis. The metabolic modulation induced by overproduction of human mutated amyloid precursor protein (APP) clearly differs between both time points. Besides the progressive perturbation of the APP interactome, functional network analysis unveiled an inverse regulation of downstream extracellular signal-regulated kinase (ERK1/2), and p38 mitogen-activated protein kinase (MAPK) routes in 2-month-old Tg2576 mice with respect to wild-type (WT) mice. In contrast, Akt and MAPK kinase 4 (SEK1)/ stress-activated protein kinase (SAPK) axis were parallel activated in the OB of 6-months-old-Tg2576 mice. Furthermore, a survival kinome profiling performed during the aging process (2-, 6-, and 18-month-old) revealed that olfactory APP overexpression leads to changes in the activation dynamics of protein kinase A (PKA), and SEK1/MKK4-SAPK/JNK between 6 and 18 months of age, when memory deficits appear and AD pathology is well established in transgenic mice. Interestingly, both olfactory pathways were differentially activated in a stage-dependent manner in human sporadic AD subjects with different neuropathological grading. Taken together, our data reflect the early impact of mutated APP on the OB molecular homeostasis, highlighting the progressive modulation of specific signaling pathways during the olfactory amyloidogenic pathology., This work was funded by grants from the Spanish Ministry
of Economy and Competitiveness (Ministerio de Economía,
Industria y Competitividad, Gobierno de España, MINECO;
Ref. SAF2014-59340-R), Department of Economic Development
from Government of Navarra (Ref. PC023-PC024, PC025,
PC081-82, PI59 and PC107-108) and Obra Social la Caixa to
ES. AG-M and KA were supported by PEJ-2014-A-61949 and
PEJ-2014-A-72151 (MINECO). ML-M and AG-M are supported
by a predoctoral fellowship from the Public University of
Navarra (UPNA). The Proteomics Unit of Navarrabiomed is a
member of Proteored, PRB3-ISCIII, and is supported by grant
PT17/0019/009, of the PE I + D + I 2013-2016 funded by ISCIII
and FEDER.

La disfunción olfatoria es un síntoma temprano y común en diversas enfermedades neurodegenerativas (ENs). Según el grado de disfunción olfatoria que presentan, existen ENs con disfunción olfatoria pronunciada, como la enfermedad de Alzheimer (EA) y la enfermedad de Parkinson (EP), y otras con déficits más leves, como ocurre en las demencias frontotemporales. Se ha sugerido que la existencia de un sustrato patológico común actuando mediante diferentes mecanismos en este amplio espectro de ENs podría ser el causante de estas diferencias en el déficit olfatorio. El bulbo olfatorio (BO) es la primera estructura del cerebro responsable de procesar la información olfatoria y el depósito de sustratos neuropatológicos como el péptido amiloide o la forma hiperfosforilada de la proteína tau en esta región se ha propuesto también como posible origen de este síntoma. Sin embargo, se desconoce si los agregados neuropatológicos son causa o consecuencia del proceso neurodegenerativo que ocurre en esta región.
En esta tesis se ha realizado un análisis molecular de alto rendimiento en BOs procedentes de: a) dos modelos animales de la EA; y b) sujetos diagnosticados con cuatro ENs incluyendo la EA, la EP, la degeneración lobar fronto-temporal con depósitos de TAR DNA-binding protein (FTLD-TDP43) y la parálisis supranuclear progresiva (PSP), con el objetivo de caracterizar los mecanismos neuropatofisiológicos que ocurren en esta región durante el proceso neurodegenerativo. Este amplio análisis ha demostrado que existe una gran alteración en la proteostasis del BO durante la EA y la EP, donde los resultados mostraron un 20% del proteoma cuantificado diferencialmente expresado. Por otra parte, en el caso de los sujetos diagnosticados con FTLD-TDP43 y PSP, el número de alteraciones fue mucho menor, constituyendo alrededor del 1% del proteoma cuantificado. Es interesante resaltar que se han encontrado tanto similitudes como diferencias en los mediadores proteicos diferencialmente expresados entre las ENs analizadas y la población control. Por otra parte, el estudio en los dos modelos animales de EA ha demostrado que, a nivel de BO, existen alteraciones moleculares previas a la aparición de placas amiloides y deficits cognitivos. Finalmente, se ha demostrado la utilidad de la proteómica dirigida a estructuras olfatorias como fuente de biomarcadores en ENs. De hecho, se propone la proteína Glucosamine-6-phosphate isomerase 2 (GNPDA2) como potencial biomarcador de la EP., It has been established that smell impairment is a common early feature of neurodegenerative diseases (NDs). In fact, there is a spectrum of olfactory dysfunction ranging from severe loss, as seen in Alzheimer’s disease (AD) and Parkinson’s disease (PD), to little olfactory deficits, as seen in frontotemporal dementias (FTD). That is why, it is likely that differential disruption of a common neuropathological substrates might be causing these differences in olfactory functionality. The olfactory bulb (OB) is the first site for the processing of olfactory information and the deposition of pathological substrates such as amyloid peptides (Aβ), α-synuclein or the hyperphosphorylated form of tau has been suggested as a potential origin of olfactory deficits. However, it remains unknown whether these protein aggregates represent a cause or consequence of the neurodegenerative process ocurring in the OB.
In this thesis, a high-throughput comparative molecular analysis of OBs derived from: a) two familial AD mouse models; and b) 4 different NDs including AD, PD, frontotemporal lobar degeneration with TAR DNA-binding protein 43 deposits (FTLD-TDP43) and progressive supranuclear palsy (PSP) subjects, has been performed in order to characterize the neuropathophysiological mechanisms occurring in this structure during the neurodegenerative process. This wide analysis has shown great disarrangements in the OB proteostasis across AD and PD stages, where 20% of the quantified proteome was differentially expressed, while a minor deregulation was observed in FTLD-TDP43 and PSP subjects, where ∼1% of the quantified proteome was affected. Interestingly, both commonalities and differences were observed in the proteomic signature across the NDs at the OB level. On the other hand, olfactory molecular disarrangements were observed at early AD stages and prior to the appearance of both Aβ plaques and memory impairments in two different mouse models. Finally, the use of olfactory proteomics as a resource for the discovery of neurodegeneration biomarkers has been demonstrated, identifying Glucosamine-6-phosphate isomerase 2 (GNPDA2) as a potential biomarker candidate for PD., Esta tesis ha contado con la financiación del MINECO (SAF2014-59340), Departamento de Salud del Gobierno de Navarra (72/2015), Departamento de Desarrollo Económico del Gobierno de Navarra, Universidad Pública de Navarra (Ayudas para la formación de Personal Investigador de la Universidad Pública de Navarra para la realización de tesis doctorales 2017), Departamento de Salud del Gobierno de Navarra (Ayudas a profesionales para la formación continuada en Ciencias de la Salud), Universidad Pública de Navarra-La Caixa (Ayudas a la movilidad de doctorandos UPNA 2016-2017-2018)., Programa de Doctorado en Ciencias de la Salud (RD 99/2011), Osasun Zientzietako Doktoretza Programa (ED 99/2011)

Alzheimer´s disease (AD) is characterized by progressive dementia, initially
presenting olfactory dysfunction. Despite the olfactory bulb (OB) is the first central
structure of the olfactory pathway, we lack a complete molecular characterization of
the transcriptional events that occurs in this olfactory area during AD progression.
To address this gap in knowledge, we have assessed the genome-wide expression
in postmortem OBs from subjects with varying degree of AD pathology. A stagedependent
deregulation of specific pathways was observed, revealing transmembrane
transport, and neuroinflammation as part of the functional modules that are disrupted
across AD grading. Potential drivers of neurodegeneration predicted by networkdriven
transcriptomics were monitored across different types of dementia, including
progressive supranuclear palsy (PSP), mixed dementia, and frontotemporal lobar
degeneration (FTLD). Epidermal growth factor receptor (EGFR) expression was
significantly increased in the OB of AD and mixed dementia subjects. Moreover,
a significant increment in the activation of signal transducer and activator of
transcription 3 (STAT3) was exclusively detected in advanced AD stages, whereas
total STAT3 levels were specifically overexpressed in mixed dementia. Furthermore,
transcription factors deregulated in the OB of mixed dementia subjects such as cAMP
Responsive Element Binding Protein 1 (CREB1) and AP-1 Transcription Factor Subunit
(c-Jun) were not differentially modulated at olfactory level across AD grading. On the
other hand, olfactory expression of this signal transducer panel was unchanged in
PSP and FTLD subjects. Taken together, this study unveils cross-disease similarities
and differences for specific signal transducers, providing mechanistic clues to the
intriguing divergence of AD pathology across proteinopathies., This work was funded by grants from the Spanish
Ministry of Economy and Competitiveness (MINECO)
(Ref. SAF2014-59340-R), Department of Economic
Development from Government of Navarra (Ref. PC025),
and Obra Social la Caixa to ES. MLM is supported by
a predoctoral fellowship from the Public University of
Navarra. The Proteomics Unit of Navarrabiomed is a
member of Proteored, PRB2-ISCIII, and is supported by
grant PT13/0001, of the PE I+D+I 2013-2016 funded by
ISCIII and FEDER.

Olfaction is often deregulated in Alzheimer’s disease (AD) patients, and is also impaired
in transgenic Tg2576 AD mice, which overexpress the Swedish mutated form of human amyloid
precursor protein (APP). However, little is known about the molecular mechanisms that accompany
the neurodegeneration of olfactory structures in aged Tg2576 mice. For that, we have applied
proteome- and transcriptome-wide approaches to probe molecular disturbances in the olfactory
bulb (OB) dissected from aged Tg2576 mice (18 months of age) as compared to those of age
matched wild-type (WT) littermates. Some over-represented biological functions were directly
relevant to neuronal homeostasis and processes of learning, cognition, and behavior. In addition
to the modulation of CAMP responsive element binding protein 1 (CREB1) and APP interactomes,
an imbalance in the functionality of the IκBα-NFκB p65 complex was observed during the aging
process in the OB of Tg2576 mice. At two months of age, the phosphorylated isoforms of olfactory
IκBα and NFκB p65 were inversely regulated in transgenic mice. However, both phosphorylated
proteins were increased at 6 months of age, while a specific drop in IκBα levels was detected in
18-month-old Tg2576 mice, suggesting a transient activation of NFκB in the OB of Tg2576 mice. Taken
together, our data provide a metabolic map of olfactory alterations in aged Tg2576 mice, reflecting the
progressive effect of APP overproduction and β-amyloid (Aβ) accumulation on the OB homeostasis
in aged stages., This work was funded by grants from the Spanish Ministry of Economy and Competitiveness
(MINECO) (Ref. SAF2014-59340-R), Department of Economic Development from Government of Navarra
(Ref. PC023-24) and Obra Social la Caixa to Enrique Santamaría. Andrea González-Morales and Karina Ausín
were supported by PEJ-2014-A-61949 and PEJ-2014-A-72151 (MINECO). Mercedes Lachén-Montes is supported
by a predoctoral fellowship from the Public University of Navarra (UPNA). The Proteomics Unit of Navarrabiomed is a member of Proteored, PRB2-ISCIII, and is supported by grant
PT13/0001, of the PE I + D + I 2013–2016 funded by ISCIII and FEDER.

Olfactory dysfunction is among the earliest features of Alzheimer’s disease (AD). Although neuropathological abnormalities have been detected in the olfactory bulb (OB), little is known about its dynamic biology. Here, OB- proteome analysis showed a stage-dependent synaptic proteostasis impairment during AD evolution. In addition to progressive modulation of tau and amyloid precursor protein (APP) interactomes, network-driven proteomics revealed an early disruption of upstream and downstream p38 MAPK pathway and a subsequent impairment of Phosphoinositide-dependent protein kinase 1 (PDK1)/Protein kinase C (PKC) signaling axis in the OB from AD subjects. Moreover, a mitochondrial imbalance was evidenced by a depletion of Prohibitin-2 (Phb2) levels and a specific decrease in the phosphorylated isoforms of Phb1 in intermediate and advanced AD stages. Interestingly, olfactory Phb subunits were also deregulated across different types of dementia. Phb2 showed a specific up-regulation in mixed dementia, while Phb1 isoforms were down-regulated in frontotemporal lobar degeneration (FTLD). However, no differences were observed in the olfactory expression of Phb subunits in progressive supranuclear palsy (PSP). To sum up, our data reflect, in part, the missing links in the biochemical understanding of olfactory dysfunction in AD, unveiling Phb complex as a differential driver of neurodegeneration at olfactory level., This work was funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (Ref. SAF2014-59340-R), Department of Economic Development from Government of Navarra (Ref. PC025), and Obra Social la Caixa to ES. AGM and KA are supported by PEJ-2014-A-61949 and PEJ-2014-A-72151 (MINECO). EPV is supported by PTA-2013/8711/I (MINECO). MLM is supported by a predoctoral fellowship from the Public University of Navarra (UPNA). The Proteomics Unit of Navarrabiomed is a member of Proteored, PRB2-ISCIII, and is supported by grant PT13/0001, of the PE I + D + I 2013-2016 funded by ISCIII and FEDER.

Glioblastoma multiforme (GBM) is the most common and aggressive type of malignant glioma. Oncolytic adenoviruses are being modified to exploit the aberrant expression of proteins in tumor cells to increase the antiglioma efficacy. E1A mutant adenovirus Delta-24-RGD (DNX-2401) has shown a favorable toxicity profile and remarkable efficacy in a first-in-human phase I clinical trial. However, the comprehensive modulation of glioma metabolism in response to Delta-24-RGD infection is poorly understood. Integrating mass spectrometry based-quantitative proteomics, physical and functional interaction data, and biochemical approaches, we conducted a cell-wide study of cytosolic, nuclear, and secreted glioma proteomes throughout the early time course of Delta-24-RGD infection. In addition to the severe proteostasis impairment detected during the first hours post-infection (hpi), Delta-24-RGD induces a transient inhibition of signal transducer and activator of transcription 3 (STAT3), and transcription factor AP-1 (c-JUN) between 3 and 10hpi, increasing the nuclear factor kappa B (NF-κB) activity at 6hpi. Furthermore, Delta-24-RGD specifically modulates the activation dynamics of protein kinase C (PKC), extracellular signal–regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK) pathways early in infection. At extracellular level, Delta-24-RGD triggers a time –dependent dynamic production of multitasking cytokines, and chemotactic factors, suggesting potential pleiotropic effects on the immune system reactivation. Taken together, these data help us to understand the mechanisms used by Delta-24-RGD to exploit glioma proteome organization. Further mining of this proteomic resource may enable design and engineering complementary adenoviral based-vectors to increase the specificity and potency against glioma., This work was funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (Ref. SAF2014-59340-R To ES), the Department of Economic Development from Government of Navarra
(ref. PC025, PC023-24, PC81-82, PI059 to ES and PI031 to JFI), the Instituto de Salud Carlos III and Fondos Feder Europeos (PI16/00066 to MMA), the Spanish Ministry of Science and Innovation (IEDI2015-00638 to MMA), the Department of Health of the Government of Navarra (to MMA), the Basque Foundation for Health Research (BIOEF, BIO13/CI/005 to MMA), Asociación Pablo Ugarte-Fuerza, Julen (to MMA). AGM was supported by PEJ-2014-A-61949 (MINECO).

Brain proteomics has become a method of choice that allows zooming-in where neuropathophysiological alterations are taking place, detecting protein mediators that might eventually be measured in cerebrospinal fluid (CSF) as potential neuropathologically derived biomarkers. Following this hypothesis, mass spectrometry-based neuroproteomics has emerged as a powerful approach to profile neural proteomes derived from brain structures and CSF in order to map the extensive protein catalog of the human brain. This chapter provides a historical perspective on the Human Brain Proteome Project (HBPP), some recommendation to the experimental design in neuroproteomic projects, and a brief description of relevant technological and computational innovations that are emerging in the neurobiology field thanks to the proteomics community. Importantly, this chapter highlights recent discoveries from the biology- and disease-oriented branch of the HBPP (B/D-HBPP) focused on spatiotemporal proteomic characterizations of mouse models of neurodegenerative diseases, elucidation of proteostatic networks in different types of dementia, the characterization of unresolved clinical phenotypes, and the discovery of novel biomarker candidates in CSF., The Proteomics Unit of Navarrabiomed is a member of Proteored, PRB3-ISCIII, and is supported by grant PT17/0019, of the PE I + D + i 2013–2016, funded by ISCIII and ERDF. Part of the work described here was funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (Ref. SAF2014-59340-R), Department of Economic Development from the Government of Navarra (Ref. PC023-PC024, PC025, PC081-82, and PI059), and Obra Social la Caixa to E.S.