Vega Laboratory - Translational Science and Molecular Medicine - College of Human Medicine - Michigan State University

Philosophy  |  Alzheimer’s  |  Biomarkers  |  Proteomics  |  Contributions  |  Grant  |  Mentoring  |  Personnel

Our cells are constantly influence by its environment. This is an important feature that allows cells to take advantage of beneficial signals or protect themselves from detrimental ones. For example, during development of the nervous system specific molecules are release to guide the formation of functional connections, while eliminating others. In contrast, a toxic molecule will trigger a defense mechanism that averts detrimental effects that could lead to cell death. These cellular responses are possible due to the capability of our proteome (all proteins in the cell) to adapt in response to environment cues. Adaptive proteome responses are responsible for the processing of signals received by molecular cues or detoxification of contaminants that could affect the cell. Disease state, then, could be described in terms of adaptive proteome responses to an, environmental or molecular, insult that affects the function of specific cells. In disease state (such as Alzheimer’s disease), the proteome changes in respond to toxic molecules that induce cellular stress. The default response to cellular stress is survival. However, if the cellular insult continues to be present in the environment, mechanisms associated with programmed cell death (i.e. apoptosis) will be activated. Therefore, the fitness of an aging brain to deal with the accumulation of environmental insults could determine the fate of an individual: disease versus health.

Adaptive proteome responses could be defined as transient changes in post-translational modification, interactions, function and/or subcellular localization. Thus, our main research interest is the identification and characterization of proteome changes associated with neurological disorders in brain tissue or body fluids (e.g. urine, blood, cerebrospinal fluid).

Alzheimer’s disease (AD) is the best known and studied dementia. AD belong to a family of neurodegenerative diseases, known as tauopathies, that are characterized by the aggregation of the microtubule-associated protein tau in the cell body of neurons. The aggregation of tau proteins is toxic to neurons. However, the process that induces tau aggregation and the corresponding cellular response that leads to neuronal death is still poorly understood. In our laboratory, we are interested in understand how neurons respond to the presence of toxic tau aggregates.

For the identification of proteome changes induced by tau pathology may provide insights about molecular mechanisms underlying tau-mediated neurodegeneration, we used the tauopathy mouse model JNPL3. JNPL3 mice express the human tau protein bearing a mutation (P301L) found in familial cases of Frontotemporal Dementia and Parkinsonism linked to chromosome 17 (FTDP17). Phenotypically, the JNPL3 hemizygous animal showed progressive motor and behavioral deficits as the mice aged, which directly correlated with the accumulation of aggregated tau proteins at specific brain regions. In other words, JNPL3 mice mimic the neurodegeneration observed in human tauopathies.

In order to identify tau-associated proteins in the course of neurodegeneration, hTauP301L proteins were immunoprecipitated from terminally ill JNPL3 mice using the human tau specific antibody Tau13 [Vega et al. (2008) J. Neurochem. 106:96]. Tandem mass spectrometric analyses (technique used to reveal the identity of proteins in a sample) of the immunoprecipitated proteins led to the identification of a novel tau-associated protein of unknown function [Vega et al. (2008) J. Neurochem. 106:96]. Sequence analysis of the identified novel protein revealed the presence of two conserved calcium-binding motifs (EF-hand motifs) and a colied-coil domain [Vega et al. (2008) J. Neurochem. 106:96]. This novel protein was named EFhd2. We demonstrated that EFhd2 is a calcium binding protein and that associates with aggregated tau. These results were validated in human tauopathy cases (AD, PSP and FTDP17) demonstrating that the association between EFhd2 and tau is not induced exclusively by the human tau expressed in the tauopathy mouse model. Therefore, EFhd2 may play an important role in the pathobiology underline tau-mediated neurodegeneration.

Why is important to study EFhd2 role in neurodegeneration?  In humans, EFHD2 gene is located in chromosome 1, specifically in a region that has been associated and linked to late-onset AD.  Importantly, we showed that EFhd2 protein level increases in AD and other tauopathies, but not in normal aging controls [Vega et al. (2008) J. Neurochem. 106:96; Ferrer-Acosta et al. (2013) J. Neurochem. 125(6):921].  Recently, we also demonstrated that EFhd2 is a novel amyloid protein able to form filaments in vitro [Ferrer-Acosta et al. (2013) J. Neurochem. 125(6):921].  Immunogold labeling and transmission electron microscopy showed that EFhd2 filaments co-aggregate with tau filaments present in the sarkosyl insoluble fraction derived from temporal cortex of AD brains [Ferrer-Acosta et al. (2013) J. Neurochem. 125(6):921].  Thus, EFhd2 is the first calcium binding amyloid protein associated to pathological tau in AD.  Currently, we are interested in characterize the pathological role that EFhd2 may play in tauopathies.

Differences in protein abundance have been reported associated with the progression of tau-mediated neurodegeneration.  We identified amphiphysin-1 (AMPH1) as a protein affected specifically in brain regions with tau pathology [De Jesús et al. (2012) NeuroReport 23:942].  AMPH1 is an essential protein in clatherin-mediated endocytosis that is involved in promoting the recruitment of Dynamin, which induces the pinch-off of recycling vesicle.  Currently, we are conducting experiments to characterize the molecular mechanisms that lead to AMPH1 protein level reduction in tau-mediated neurodegeneration.

Interestingly, AMPH1 has been directly implicated in a rare neurological disorder known as stiff-person syndrome (SPS). SPS is classified as an autoimmune disease divided in two groups: those with anti-glumatic acid decarboxylase (GAD) antibodies and others with auto-AMPH1 antibodies. Although the pathophysiological process that leads to the generation of anti-AMPH1 antibody positive SPS is still unknown, it is tempting to speculate that AMPH1 protein reduction may lead to the secretion of AMPH1 peptides that triggers an autoimmune response. Therefore, we set to detect auto-AMPH1 antibodies using recombinant GST-tagged AMPH1 (100 ng) as target or “bait.” Auto-AMPH1 antibodies were detected in the serum obtained from JNPL3 mice but not control (non-transgenic) mice. The results obtained so far indicate that increased in the titer of auto-AMPH1 antibodies is associated with AMPH1 protein reduction and tau-mediated neurodegeneration in the tauopathy mouse model JNPL3 (Nogueras-Ortiz et al. (2014) Front Neurosci. 7:277). Thus, auto-AMPH1 antibodies could be considered a biomarker of tau-mediated neurodegeneration with the potential to become a molecular diagnostic tool. Experiments are being conducted to validate the detection of auto-AMPH1 antibodies associated with neurodegeneration in human tauopathy cases.

Furthermore, we are interested in the understanding of brain region specific vulnerability in Alzheimer’s disease. Neuropathological analyses of familial (i.e. inherited mutation in either APP, PS1 or PS2 genes) and sporadic (i.e. no inherited) tauopathies showed similar brain region specific vulnerability. Therefore, in addition to the vulnerability of some brain regions, there are other brain regions resistant to toxic tau molecules. A proteomic approach is being conducted to identify differences in the proteome between vulnerable and resistant brain regions to tau-mediated neurodegeneration. The results obtained will provide insights into the molecular mechanisms that may contribute to tau-toxicity resistance in specific brain regions as well as identify other proteins associated with tau-mediated neurodegeneration in vulnerable brain regions.

The proteomics approach to understand adaptive proteome responses associated with disease state could be used for the study of different neurological disorders. Autism is a multi-factorial disorder that is poorly understood. In Autism spectrum disorder, there are not identified molecular biomarkers that facilitate the diagnosis or understanding of the pathobiology of this disorder. Despite several it has been discussed the importance of using a proteomics approach for the study of Autism, at the present time there are not published results using this kind of research. A proteomics approach that takes in consideration different ethnic backgrounds, treatments or intervention, and selection/preparation of specific body fluids (e.g. urine, serum, cereblospinal fluid) will be essential to contribute to the understanding and diagnosis of Autism. Therefore, our expertise in mass-spectrometry based proteomics for the identification of biomarkers associated with neurodegeneration will be easily applied for the development of diagnostic tools and understanding of the pathobiology of this important scientific conundrum that afflicts our children.

Identification and characterization of novel tau-associated proteins. As PI, my research team was the first one to show that EFhd2 is associated with pathological tau species [1]. We were able to demonstrate that EFhd2 is a novel amyloid protein that self-oligomerize and co-localize with aggregated tau in the somatodendritic compartment of Alzheimer’s disease brain [1-4]. Currently, we are conducting experiments to characterize the role of EFhd2 in Alzheimer’s disease and related tauopathies.

1. Vega IE, Traverso EE, Ferrer-Acosta Y, Matos E, Colon M, Gonzalez J, Dickson D, Hutton M, Lewis J and Yen SH. A novel calcium binding protein is associated with tau proteins in tauopathy. J. Neurochem. (2008) 106(1):96-106. 
2. Ferrer-Acosta Y, Rodriguez-Cruz EN, Vaquer-Alicea AC and Vega IE (2013) Functional and Structural Analysis of the Conserved EFhd2 Protein. Protein Pept Lett. 20:573-583.
3. Ferrer-Acosta Y, Rodriguez-Cruz EN, Orange F, De Jesus-Cortes H, Madera B, Vaquer-Alicea J, Ballester J, Guinel M JF, Bloom GS, Vega IE (2013) EFhd2 is a novel amyloid protein associated to pathological tau in Alzheimer’s disease. J. Neurochem. 125:921-931.
4. Vazquez-Rosa E, Rodriguez-Cruz EN, Serrano S, Rodriguez-Laureano L, Vega IE (2014) Cdk5 phosphorylation of EFhd2 at S74 affects its calcium binding activity. Protein Science 23:1197-207.

DNA repair and neurite outgrowth. At the outset of my research career, I contributed to the characterization of proteins involved in DNA repair and neurite outgrowth. First, I worked on the functional characterization of the association of the DNA repair protein Rad23 with the proteasome. My contribution was the construction of Rad23 truncation mutants and the development of experiments that demonstrated the co-immunoprecipitation of Rad23 with functional proteasomes. The results established a direct association between DNA repair and the ubiquitin-proteasome pathway. Then, I applied the molecular and biochemical techniques learned to characterize the role of the Exocyst complex and septins in neuronal development. I was able to demonstrate that Exocyst and septins are directly involved in neurite outgrowth. These published results are used by others to further the molecular characterization of DNA repair and neuronal development in health and disease.

1. Schauber C, Chen L, Tongaonkar P, Vega I, Lamberstson, D., Potts, W. and Madura, K. (1998) Rad23 links DNA repair to the ubiquitin-proteasome pathway. Nature 391: 715-718.

2. Vega IE and Hsu SC (2001) The Exocyst complex associates with microtubules to mediate vesicle targeting and neurite outgrowth. J. Neuroscience 21: 3839-3848.

3. Vega IE and Hsu SC (2003) The septin Nedd5 protein associates with both the Exocyst complex and microtubules and disruption of its GTPase activity promotes aberrant neurite outgrowth in PC12 cells.  NeuroReport 14: 31-37.

Pathological tau characterization.  Tau aggregation is a central pathological hallmark in Alzheimer’s disease and related disorders.  Therefore, the characterization of tau aggregates and mechanisms that mediate its aggregation is important to gain insights that lead to the development of therapeutic strategies. During my postdoctoral training, I contributed (using a proteomics approach) to identify novel tyrosine phosphorylation sites on tau proteins and correlated them to the accumulation of aggregated tau in a tauopathy mouse model and Alzheimer’s disease brain.  Tyrosine phosphorylation of tau proteins is currently studied as an important molecular signature of the progression of tau-mediated neurodegeneration.

1. Sahara N, Vega IE, Ishizawa T, Lewis J, McGowan E, Hutton M, Dickson D and Yen SH  (2004) Phosphorylated p38MAPK specific antibodies cross-reacted with sarkosyl-insoluble hyperphosphorylated tau proteins.  J. Neurochem. 90: 829-838.
2. Vega IE, Cui L, Propst JA, Lee G, Hutton M and Yen SH (2005) Increase in tau tyrosine phosphorylation correlated with the formation of tau-aggregates.  Mol. Brain Res. 138:135-144 
3. Ko L-W, DeTure M, Sahara N, Chihab R, Vega IE and Yen S-H  (2005)  Recent advances in experimental modeling of the assembly of tau filaments.  Biochim. Biophys. Acta 1739: 125-139.

Biomarker identification and characterization. The understanding of adaptive proteome responses in tauopathy is an area that I also contributed.  As PI, I led a group of students in the study of the effect that the expression of human tau mutant has on the proteome of brain regions known to be vulnerable versus resistant to tauopathy.  This brain region specific proteomics approach led to the identification of AMPH1, as a protein whose abundance is reduced, specifically, in brain regions affected by tau-mediated neurodegeneration.  This result was validated in human tauopathy cases.  AMPH1 is a protein involved in clathrin-mediated neurodegeneration and associated to BIN1, a known risk factor of Alzheimer’s disease.  Moreover, we showed that auto-antibodies against AMPH1 are detected in the serum of a tauopathy mouse model, which correlated with the accumulation of aggregated tau.  These results demonstrated a connection of relevant mechanisms associated with tauopathy, namely endocystosis and immune respond.  It is important to point out that this research work was done by undergraduate and graduate students under my supervision while I was at the University of Puerto Rico.  Now at Michigan State University, we are working to define the molecular mechanisms that lead to tau-mediated reduction of AMPH1 protein levels and generation of auto-immune antibodies in tauopathy.  

1. De Jesús-Crotés H, Nogueras-Ortíz CJ, Gearing M, Arnold SE and Vega IE (2012) Amphiphysin-1 protein level changes associated with tau-mediated neurodegeneration.  Neuroreport. 23:942-946
2. Nogueras-Ortiz CJ, De Jesús-Cortes HJ, Vaquer-Alicea J, Vega IE. (2014)  Novel autoimmune response in a tauopathy mouse model.  Front Neurosci. 2014 Jan 10;7:277. doi: 10.3389/fnins.2013.00277. eCollection 2014 Jan 10.

Ethnicity and neurodegeneration.  An important aspect in the understanding of the etiology of Alzhemier’s disease (AD) is to study the progression of this disease in the population.  AD is a multifactorial and multigenic disease that shows differentially presentation based on ethnicity.  As PI, I contributed to the characterization and profiling of AD in the Latino population.  The results contributed to understand the effect of ethnicity and genetic makeup in the symptomatology of AD. 

1. Figueroa R, Steenland K, MacNeil J, Levey AI and Vega IE (2008) Geographical differences in the occurrence of Alzheimer’s disease mortality: United States vs. Puerto Rico. Am J Alzheimer Dis Other Demen  23:462-469  
2. Livney MG, Clark CM, Karlawish JH, Cartmell S, Vega IE, Entenza-Cabrera F and Arnold SE (2011) Ethnoracial differences in the clinical presentation of Alzheimer’s disease. Am. J. Geriatr. Psychiatr. 19:430-439.
3. Arnold SE, Vega IE, Karlawish JH, Wolk DA, Nunez J, Negron M, Xie SX, Wang LS, Dubroff JG, McCarty-Wood E, Trojanowski JQ and Van Deerlin V. (2013) PubMed Abstract: Frequency and Clinicopathological Characteristics of Presenilin 1 Gly206Ala Mutation in Puerto Rican Hispanics with Dementia.  J Alzheimers Dis. 33:1089-1095

Proteomics and collaborative skills.  My expertise in proteomics analysis allowed me to contribute to diverse areas of research, from marine to food science.  For example, I contributed to demonstrate that the molecular mechanism that barnacles use to generate the cement that allows them to attach to surfaces under water is similar to human blood coagulation.  This result indicated a conserved survival mechanism between barnacles and humans, as well as established barnacles as anti-coagulating drug testing model.  As PI, I also contributed to demonstrate that garlic extract modified human beta-hemoglobin.  We identified the specific molecule in garlic extract and the site of modification on beta-hemoglobin.  Also, we contributed to the identification of specific post-translational modifications of proteins expressed in bacteria and yeast that are involved in lipid metabolism and RNA degradation, respectively.  The opportunity to successfully contribute to different fields of research demonstrates my flexibility, technical adaptation and collaborative skills.  I am confident that these skills will facilitate the advancement of my research career at Michigan State University.    

1. Dickinson GH, Vega IE, Walh KJ, Orihuela B, Beyley V, Rodriguez EN, Everett RK, Bonaventura J and Rittschof D (2009) Barnacle cement: a polymerization model based on evolutionary concepts. J Exp. Biol. 212:3499-3510
   Lab Spaces Press Release: Super sticky barnacle glue cures like blood clots; BBC News Article: Barnacles' sticky secret revealed

2. Bonaventura J, Rodriguez EN, Beyley V, and Vega IE  (2010) Allylation of intraerythocytic deoxygnated hemoglobin by raw garlic extracts.  J. Med Food 13:943-949. 
3. Trujillo U, Vázquez-Rosa E, Oyola-Robles D, Stagg LJ, Vassallo DA, Vega IE, Arnold ST, Baerga-Ortiz A (2013) Solution structure of the tandem acyl carrier protein domains from a polyunsaturated fatty acid synthase reveals beads-on-a-string configuration. PLoS ONE 8(2):e57859.
4. Lasalde C, Rivera A, Leon A, Gonzalez J, Estrella L, Correa M, Cajigas I, Bracho D, Vega IE, Wilkinson M, Gonzales CI (2014) Identification and Functional Significance of Novel Phosphorylation Sites in the NMD Protein Upf1.  Nucleic Acid Research 42:1916-1929.

Complete publication list may be found at:
Google Scholar: http://scholar.google.com/citations?user=WMjeD3cAAAAJ&hl=es
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/?term=Vega+IE

Current Grant Support: NIH-NINDS 7R15NS081593

Active Research Support

Thorek Memorial Foundation         Lipton/Vega (Multi PI)           01/01/2017-12/31/2019

A High Throughput Process to Study Interacting Genetic Risk Factors in Disease through Informatics and Gene Modification

This grant is to develop a combinatorial gene manipulation and functional screen (COGMAS) utilizing genomic data informed by functional network analyses.

Role: PI

Effort: 20% AY

P30AG053760                                   Paulson (PI)                            08/01/2016-07/31/2021          

Michigan Alzheimer’s Disease Core Center

The MADC will support a wide range of studies on Alzheimer’s disease and other dementias while also educating scientists, health care professionals and the public on the causes and treatment of dementias in the state of Michigan.

Role: Co-I

Effort: 5% AY

T32NS007222                                                Feldman E (PI)                       7/01/2017-6/30/2022

T32-Training in Clinical and Basic Neuroscience

This training program in disease-oriented neuroscience trains physicians and basic scientists to conduct basic and clinical disease-oriented neuroscience. We offer basic science and translational and clinical science training in neurodegenerative disorders, neuromuscular diseases, sleep disorders, stroke, and systems neuroscience.

Role: Co-I

Effort: 15% AN (summer salary)

MADC-Pilot Project Grant              Vega (PI)                    05/01/2017-04/30/2018          

P30AG053760 Paulson (PI)

Determine the role of the novel efhd2 protein on tau oligomerization

Role: PI

Effort: 5%

relinquished Research Support

1R25GM097635-06               Vega (PI)                    09/01/2015-08/31/2020          

NIH/NIGMS

Neuroscience Research Opportunities to Increase Diversity (NeuroID)

This a competitive renewal of an undergraduate training program known as NeuroID.  NeuroID aims to increase the opportunities available for undergraduate students in the area of Neurosciences.  To achieve this goal, we proposed to continue and enhanced the established multi-component program that includes research experience, enhanced academic training, community outreach and professional development activities.

Role: The grant was relinquished to the University of Puerto Rico upon relocation to Michigan State University.

Completed Research Support

MSU S3 Collaborative Grant          Vega (PI)                    01/01/2017-12/31/2017

Health disparities in Alzheimer’s disease: a community-based participatory research approach

Role: PI

Effort: N/A

http://s3.msu.edu/funded-projects-2016-2017/

1R15NS081593-01                 Vega (PI)                    12/01/2012-11/30/2015          

NIH/NINDS

Autoimmune Biomarker Profiling In Tauopathy

The development of non-invasive diagnostic tools for neurodegenerative disorders, such as Alzheimer's disease and other tauopathies, is crucial for the testing of new forms of treatment that may prevent, retard or revert the progression of these diseases. The proposed research plan intends to characterize and validate the identification of autoimmune responses in the course of tau-mediated neurodegeneration. The results obtained may lead to the discovery of disease-specific protein biomarkers that serve as diagnostic tools and to better understand the pathobiology of tauopathies.

Role: PI

1R25GM097635-01   Garcia-Arraras/Vega (Multi-PIs)   09/01/2010-08/31/2015               

NIH/NIGMS

Neuroscience Research Opportunities to Increase Diversity (NeuroID)

NeuroID aims to increase the opportunities available for undergraduate students in the area of Neurosciences.  To achieve this goal, it is proposed a multi-component program which would include research experience, enhanced academic training, community outreach and professional development activities.

Role: PI

1SC1NS066988-01    Vega (PI)                    08/01/2009-12/31/2013                      

NIH/NINDS

The role of a novel tau-associated protein in neurodegeneration

The proposed project intends to characterize the role that the novel tau-associated protein TEA plays in the development and/or progression of tau-mediated neurodegeneration.

Role: PI

Mentor-Mentee Relationship: a two way street

Respect and humbleness are key factors in the mentor-mentee relationship that opens an important component of the learning process, communication.  Mentors should guide mentees through the path of a research career, pointing out the important hallmarks and milestones but allow them to discover the inners confinements of science.  

Respect: It is very important to establish the expectations from the very beginning; not only what the mentor expects from the mentee, but also what the mentee should expect from the mentor, including how controversies or misunderstandings are going to be dealt with.  You should request from the mentee the same that you, as mentor, are willing to give; the same level of commitment, responsibility and readiness is required from both, the mentor and mentee.  The mentor should also level the playing field, establishing that there are different responsibilities that need to be fulfilled.  The mentee needs to understand that they have to work hard to achieve their goals, but that evaluations will be based on a fair process.  Once the rules are established, the rest is to just to be respectful to each other…

Humbleness: How you fill a cup that is already full?  The only way is to empty it first.  Both cups (mentor and mentee) need to have space for new knowledge.  The mentor should be opened to learn from the mentee and the mentee should not think that the mentor knows everything and cannot make mistakes.  A conflictive or authoritarian relationship will interfere with the learning process.  Thus, it is very important that both parts on this learning equation approach their relationship eager to use their critical thinking skills and to gain the most knowledge from each other as possible.    

Mentor’s and Mentee’s responsibilities: what to expect?

All lab members: I expect everyone to be respectful, professional, honest and responsible. I also expect that you feel free to express your ideas, suggestions and concerns. Importantly, everyone need to show the highest level of scientific integrity, work hard, carefully design of experiments to maximize efficiency and be passionate at all times. No be afraid of made mistakes, but be afraid of not having the necessary humbleness to accept them and learn from them.

Since I expect that we will made mistakes or fail at some point, it is important to encourage and support your colleagues. I expect that we work as a team, where everyone supports each other efforts. Respect each other space and follow the “25 rules for a healthy laboratory environment.”

Mentor: You should expect from me to organize and manage laboratory resources, fairly and consistently assess mentees’ learning and research progress, provide honest and practical feedback, maintain a professional environment, provide openness for discussion and exchange of ideas, promote and encourage career development activities, facilitate networking with scientists in the field.  You should be confident that I will support graduate students in their program requirements and to achieve all the necessary career milestones.  I will support and facilitate the Post-docs’ career development.  I will encourage you to travel, nominate you for awards and be an advocate of your work and career.  I assure you that if you ask for help and I can’t provide it, I will do all my best to find someone that can help you.  Your success is my success! 

Mentee: I expect from Graduate Students and Post-doctoral Fellows the highest level of scientific integrity and ethics.  I expect that you follow established protocols, contribute to scientific discussion, keep up with the literature, be on-time and prepare for lab meetings and have an open disposition to collaborate with every lab member in the lab.  Importantly, your lab notebook needs to be organized, clean and up-to-date at all times. (Click to read the Lab Notebook Guide.)  I will not accept any behavior that diminishes or undermines a productive and professional environment in the lab.  I expect you to take ownership of your career development and work hard to achieve your goals.   

Authorship: I define authorship as the intellectual and technical contribution to a research project.  The order of authors in a manuscript will be assigned base on the level of contribution of each individual.  In other words, if you want to be an author on a manuscript, you have to significantly contribute to its development.  

Conflict or Problem Resolutions: As in any relationship, there are may be times when conflict or problems are unavoidable.  First, regardless of the magnitude of the situation, I always expect a professional behavior.  I will not tolerate any yelling, cursing or violence among members of the lab.  Second, I will serve as mediator, meeting with each party individually and then as a group.  In a conflict or problem that involves me (i.e. Irving Vega) an external mediator will be selected from faculty members of the Department or Thesis Committee Members. The decision reached is final and set the end of further discussions about the subject.