The problem

Aging is the main factor contributing towards both Parkinson's (PD) and Alzheimer's (AD) diseases. These chronic diseases are incurable and their disabling effects may continue for years or even decades.

Studies on animal models of AD and PD and on human postmortem brain tissues, indicate that many pathological changes in the brain derive from a network of local stresses, like oxidative stress, tightly connected to inflammatory and proteotoxic stresses. Local stressful conditions are probably challenged by pathologically modified proteins, and, through a vicious cycle, may further trigger alteration of key molecules.  

Our team has been studying protective mechanisms used to maintain homeostatic respones and how these mechanims coudl be targeted pharmacologically to provide superior defense.

Our approach

We are currently studying the role of transcription factor NRF2 in protection against stimuli that induce neurodegeneration.

NRF2 is a protein that regulates the expression of about 250 genes. These genes possess the antioxidant response element (ARE) in their promoters. The genes participate in adaptive responses to oxidative, inflammatory and proteotoxic stress and in the regulation of enzymes involved in biotransformation and glutathione metabolism.

Using genetically modified rodent models as well as pharmacological approaches, we are studying the contribution of this transcription factor to the protection against oxidative damage and neuroinflammation in toxic (MPTP and 6-OHDA) and genetic (alpha-synuclein) models of Parkinson's disease and in transgenic mice possessing amyloidopathy (APPV717I) and tauopathy (TauP301L) , which are characteristic of Alzheimer's disease.


Generation of knowledge: Understanding the mechanisms that regulate NRF2 is fundamental to determine its physiological role and its pathological alterations as well as to design new pharmacological strategies. We are currently studying the regulation of NRF2 by signaling pathways. We have already described its regulation by the GSK-3/beta-TrCP pathway. We are now analyzing its participation in cell signaling by primary cilium and proliferative stimuli.

Low-grade chronic inflammation is a key element of neurodegenerative diseases. We are studying the crosstalk between NF-kB and NRF2, key elements in the pro and anti-inflammatory phenotypes of microglia.

Applicability: in collaboration with several companies, we are looking for novel mechanisms of regulation of NRF2 in the brain that could serve to reinforce its activity against neurotoxic stimuli. In preclinical models of Parkinson's disease, we are focusing on repurposing of dimethyl fumarate, a compound already used in clinical practice for multiple sclerosis.

Ongoing research projects

Title: NRF2: biomarcador y evaluación como diana terapéutica para la Esclerosis Lateral Amiotrófica
Length: 01/01/2019-31/12/2020.
Funding agency: Fundación Tatiana Pérez de Guzmán el Bueno
Principal investigator: Ana I. Rojo
Summary: There is an urgent need for risk-predictive, early prognosis/monitoring biomarkers and disease modifying strategies for Amyotrophic Lateral Sclerosis (ALS). In recent years transcription factor NRF2 is emerging as a novel target for other neurodegenerative disorders. NRF2 controls the expression of over 250 genes involved in multiple homeostatic functions and reverses multiple genetically validated ALS pathophenotypes such as oxidative, inflammatory, mitochondrial and metabolic alterations as well as proteinopathy. We have reported in over 20 articles of D1 decile and Q1 percentiles that pharmacological activation of NRF2 provides a disease modifying benefit in several models of neurodegenerative diseases but its relevance as biomarker and disease modifier has been hardly evaluated for ALS. In this project we will address the relevance of NRF2 in ALS with three high impact objectives: 1) preclinical validation of NRF2 selective drugs in highly standardized models of motor neurons/astrocytes derived from IPSCs of ALS patients, cell cultures and mice submitted to cerebrospinal fluid (CSF) of ALS patients and highly validated genetic mouse models of ALS. For this purpose, we will use, as proof-of concept, dimethyl fumarate (DMF), a dru already in use for therapy in multiple sclerosis (MS) that targets NRF2 in the brain. 2) On the clinical side, we will identify predictive biomarkers based on the analysis of the NRF2 transcriptional signature in white blood cells of ALS patients. 3) Those studies will reinforce current and future interactions with the biopharmaceutical industry. Our team, composed by a combination of basic neuroscientists and clinical neurologists, will provide new directions to identify novel early biomarkers of ALS, and will establish NRF2 as a novel therapeutic target in preclinical models, paving the way for industry-sponsored clinical trials. 

Title: Knowledge transfer in redox biology for developing advanced molecular tools in neurodegenerative diseases – focus on the signature of NRF2 transcription factor in diagnosis and therapy.
Funding agency: Competitiveness Operational Programme (COP). European Union. Code:  P_37_732
Principal investigator: Antonio Cuadrado
Summary: Studies on animal models of AD and on human postmortem brain tissues, have indicated that many pathological changes in the AD brain derive from a network of local stresses, like oxidative stress , tightly connected to inflammatory and proteotoxic stresses. Brain inflammation has a certain echo in periphery. Blood biomarkers may be very reliable for characterizing drug interactions with their target, pharmacologic mechanistic response, and off-target or adverse effects. In close relation to inflammation, enhanced oxidative stress was evidenced in AD, but conventional antioxidant therapies proved limited efficacy in clinical trials . Therefore, more targeted therapies focused on molecular components of the endogenous antioxidant system and related mechanisms are currently investigated in clinical trials. In this respect, a promising therapeutic approach in AD, as well as in other neurodegenerative disorders with altered oxidative status and low-grade chronic inflammation, is the activation of the transcription factor Nrf2 . Several activators of Nrf2 are currently on the market or are investigated in clinical trials. More details on Nrf2 biology in health and disease are provided in the Impact study. So far results indicate that activation of Nrf2 may be more successful for preventing progression of early stage pathologies than for reversing advanced pathological conditions.
To monitor the progress of this project see ResarchGate

Title: Developing preclinical and clinical biomarkers of NRF2 pathway activation for therapeutic application in neurodegenerative diseases
Funding agency: Joint Programme for Neurodegenerative Diseases (JPND-COEN 2017-18)
Principal investigator od teh Spanish partner: Antonio Cuadrado
Summary: There is an urgent need for effective neuroprotective therapies for neurodegenerative disorders including motor neuron disease and Alzheimer’s disease. The heterogeneity and complexity of these disorders have proved major challenges for therapy development. However, in both disorders neuronal injury involves a combination of age-related attenuation of protective responses; oxidative stress; mitochondrial dysfunction; dysregulation of proteostasis and neuroinflammation. In the presence of such cellular stresses, an up-regulation of the NRF2 signalling pathway increases the expression of an array of cytoprotective genes. This response is attenuated during ageing and also in MND and AD, as well as models of these disorders. Compelling evidence has emerged from robust model systems, with major contributions from the two teams underpinning this application, that NRF2 activation has positive benefits in protecting neurons from injury in MND and AD. The overall objectives are to develop multiple biochemical, transcriptomic and imaging biomarkers in robust animal models of MND and AD and linked with evaluation in human biosamples and imaging studies, to determine target engagement and indices of therapeutic efficacy of two NRF2-activating compounds (S-apomorphine and dimethyl fumarate) for future translation into human experimental medicine studies. Such ‘translational biomarkers’ are regarded as critical parameters for successful drug development.

Títle: Role of NRF2 in the function and fate of the brain with Alzheimer's. (Universidad Autónoma de Madrid).
Funding agency: Programa Retos los Retos de la Sociedad, Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016. MINECO. Code: SAF2016-76520-R
Length: 01/01/2017-31/12/2019.
Principal investigtor: Antonio Cuadrado
Summary: Neurodegenerative diseases such as Alzheimer’s disease (AD) represent the most important societal challenge to balance health span with life span and the WHO’s impulsed Global Action Against Dementia, has stated the aim of finding a disease modifying treatment for AD by year 2025. With this in mind, thanks to national and international grants, collaborations with biopharmaceutical companies, and several patents, out team has been pioneer in studying the role of transcription factor NRF2 (Nuclear factor (erythroid-derived 2)-like 2) in preclinical mouse models and postmortem samples of patients with Parkinson’s disease and with AD and have become an international reference in this field with over 35 articles published in the past five years on this topic, many of them in international journals of the first decile and quartile. Initially known for its defensive response against oxidative stress, nowadays NRF2 is recognized to regulate up to 1% of human genes providing global homeostatic responses to multiple stimuli such as oxidant, inflammatory, metabolic and proteotoxic stress, all of which are crucial elements in AD pathology, its risk factors and its comorbidities. NRF2 has a tremendous impact on physiology and pathology and therefore it is very tightly regulated, mainly at the level of protein stability. In addition to the well-known regulation by the ubiquitin E3 ligase adapter KEAP1, which links NRF2 to biotransformation and redox metabolism, we have identified a novel mechanism based on signaling pathways that regulate glycogen synthase kinase-3 (GSK-3), a crucial kinase involved in TAU phosphorylation. This finding allows us to remain at the front of the characterization of signaling pathways that impinge on NRF2. The team, will explore innovative aspects of NRF2 pathophysiology in tauopathies and AD along the following areas: 1) Novel mechanisms of regulation of NRF2 transcriptional activity in connection with endosomal trafficking, 2) Impact of NRF2 in the pathophysiology of AD with focuses on M1/M2 polarization of microglia in response to fractalkine signaling, chaperone mediated autophagy of TAU protein and modulation of the neural stem cells from the subgranular zone of the hippocampus in our PROPRIETARY MOUSE MODEL of AD based on expression of APP(V717I) and TAU(P301L) mutant proteins in a genetic background with and without NRF2, 3) Further characterization of the best NRF2 activator, already in clinical use for multiple sclerosis, dimetyl fumarate (DMF), in our preclinical AD mouse models and characterization of KEAP1-independent regulation of NRF2 and epigenetic changes induced by the DMF/NRF2 response. 4) Analysis of the NRF2 transcriptional signature in serum and white blood cells of mild cognitive impairment and AD patients in search for innovative biomarkers of disease monitoring. We hope that this project will make a significant contribution to open an innovative strategy for a disease modifying therapy of AD.

Títle: Glial dysfunction in Alzheimer’s disease: pathologic implications and clinical potential 
Funding agency: Intramural colaborative project of Ciber sobre enfermedades neurodegenerativas (CIBERNED)
Coordinator: Javier Vitorica (US) // Joan Comella (UAB) // Javier Saes-Valero (UMH) // Maria Antonia Gutierrez (UM) // Antonio Cuadrado (UAM)
Length: 01/1/2018-31/12/2019
Summary: The present project proposes a new pathogenic scenario in Alzheimer's disease. We propose the microglial dysfunction and consequent astroglial hyperactivation as a fundamental mechanism in the neurodegenerative process. This new scenario could give answers to the continuous failure in the translation of the studies from models to the human clinic. Therefore, it highlights the need to reproduce this dysregulation of the innate cerebral immune response into the animal models to increase their predictive value and to assure a greater success of the therapies in humans.
Although microglia may play a cytotoxic role in neurodegenerative pathologies, the role in Alzheimer's is not well characterized. Recent results by the consortium members demonstrate that the microglial cells undergo a degenerative process in the hippocampus of Braak V-VI patients. This degeneration totally opposites of that observed in the Abeta producing models, classically used for preclinical studies. Consequently, as first objective, we propose to evaluate the impact on the Alzheimer's pathology of the microglial dysfunction observed in patients. These data would improve the knowledge of the pathology and, on the other hand, it will help to improve the current murine models. On the other hand, astroglia is considered a neuroprotective cell, based on its normal (nonpathological) function in trophic support, redox homeostasis, elimination of toxic molecules, vascularization and control of synaptic activity. However, the astroglial specific role in the development of Alzheimer's disease is unknown. It is well known that astrocytes are activated around the Abeta plaques. However, their phenotypic changes and gene expression patterns are not known in detail. Also, it is unknown how the factors secreted by active astrocytes may contribute to the development of the pathology. In this project we propose to approach this problem by evaluating, on the one hand, the repercussion of microglial deficiency on the phenotype of astrocytes and, on the other hand, the role played by the transcription factor Nrf2 as the master regulator of several hemostatic cytoprotective mechanisms.
Finally, and based on the results obtained in the previous aims, we propose the characterization of the possible microglial and / or astroglial "signature" in collections of cerebrospinal fluid (CSF) of subjects with mild amnestic cognitive impairment (prodromic stages), up to subjects with Alzheimer's disease. On the other hand, the most relevant results obtained in the first two objectives will be confirmed in post mortem samples of patients, available by several members of the consortium.

Títle: Development of new NRF2-activating drugs for innovative therapiy of Alzheimer's disease
Funding agency: Autonomous Community of Madrid (Spain)
Coordinator: Manuela G. García (UAM) // María Isabel Rodriguez (CSIC) // Antonio Cuadrado (UAM)
Length: 01/1/2018-31/12/2019
Summary. The latest drug introduced in the clinic for the palliative treatment of Alzheimer's disease (AD) was in 2003. Since then, a great human and economic effort has been made to achieve more effective but unsuccessful treatments. One of the possible causes of the failure of these drugs may be that the targets (mostly focused on reducing Abeta pathology) have not been the adequate. Consequently, the search for new effective therapeutic strategies for the treatment of Alzheimer's disease (AD) is a priority. This multidisciplinary project, composed of medical chemists, molecular biologists, pharmacologists and physicians, aims to search for an non-conventional drug for AD. For this, we propose as target the transcription factor NRF2. NRF2 is a master regulator of several hemostatic mechanisms that are altered in AD, such as redox status, neuroinflammatory response, protease and autophagy, hence the chances of success may be higher than those developed so far , such as the prevention of amyloid beta accumulation, TAU hyperphosphorylation or the regulation of neurotransmitter release. To develop non-conventional therapies for AD based on the enhancement of endogenous defenses through the induction of NRF2, we will follow several strategies, ranging from the virtual screening of chemical libraries to the optimization of multi-target compounds that activate NRF2 in which we already have experience and patents. Both the compounds selected from virtual screening and those synthesized in the optimization programs will undergo in vitro pharmacological screening structured at several levels, which will allow the identification of the compounds with the best pharmacodynamic and toxicological profiles to be evaluated in in vivo models relevant for EA pathology The expected results of this project can be summarized as follows: (i) To identify new NRF2 activators inhibiting NRF2/beta-TrCP interaction by virtual screening and subsequent cellular screening, (ii) Design and synthesize four families of NRF2 activators with multitarget activity that reduce oxidative stress, neuroinflammation and improve autophagic systems to eliminate accumulation of protein aggregates in order to promote neuroprotection in in vitro and in vivo models related to AD, (iii) Obtain a compound from each family with a suitable ADME profile and low toxicity; (iv) Demonstrate oral therapeutic efficacy of one compound from each family in an in vivo model of AD and (v) to protect the intellectual property of the project, we expect to deposit three or four Spanish patents with option to their PCT extension. The scientific impact is linked to the proof of concept of a new target NRF2 for the development of new drugs for the treatment of AD that could be extended to other neurodegenerative diseases. Obtaining an effective treatment for AD implies a socioeconomic impact on two levels: one, by improving the quality of life of patients and caregivers and, the other, in savings in the National Health Systems.: 



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