LABORATORY OF CELLULAR STRESS AND BIOMEDICINE

Programmed cell death is essential for the development and maintenance of cellular homeostasis and its deregulation results in a variety of pathological conditions. The BCL-2 family of proteins is a group of evolutionary conserved regulators of cell death that operate at the mitochondrial membrane to control caspase activation. This family is comprised by both anti-apoptotic and pro-apoptotic members, where a subset of pro-apoptotic members, called 'BH3-only' proteins, act as upstream activators of the core pro-apoptotic pathway (Fig. 1).  In addition to their known role at the mitochondria, different BCL-2-related proteins are located to the endoplasmic reticulum (ER) membrane, where new functions have been recently proposed by our laboratory. We are focused in studying the role of BCL-2 protein family on stress responses associated with pathological conditions affecting the nervous system and its association with organelle stress.
Alterations in organelle function have devastating consequences for the proper function of the cell. Stress injuries initiate multiple signaling responses, either to adapt to the new conditions or to activate specific cell death pathways, if a critical threshold of damage has been reached. Our laboratory is committed to the study of cellular strategies involved in adaptation to chronic organelle damage, which are linked to several neurological disorders. The endoplasmic reticulum (ER) has important cellular functions and is well known for its role as a sophisticated machinery for protein folding and secretion. Alterations on ER function lead to the accumulation of unfolded proteins in its lumen, a cellular condition termed “ER stress”. ER stress triggers a complex adaptive reaction known as the “Unfolded Protein Response” (UPR), which aims to restore this organelle’s homeostasis. Sustained ER stress ultimately promotes apoptosis, where the members of the BCL-2 family of proteins are essential in the initiation of cell death. Nevertheless, the mechanisms that control the transition from an adaptive state to cell death processes remain unknown.
 

How do cells sense ER stress? ER stress stimulates distinct  stress sensor signaling pathways including the IRE1a. IRE1a is a ER transmembrane protein that transduces stress signals to the nucleus by controlling the expression of the transcription factor X-box-binding protein 1 (XBP-1). XBP-1 is one of the most important players in the adaptation to ER stress conditions because it mediates the upregulation of crucial UPR-related genes involved in folding, secretion, ER biogenesis, protein quality control and many other functions. The mechanisms underlying IRE1a activation remain poorly understood. We have described recently a new function for some BCL-2 family members in the signaling through a direct interaction due to the formation of control of IRE1a  a protein complex. In the current project we propose to further study the cellular function and regulation of IRE1a.
ER stress and diseases - Chronic ER stress is associated with neurodegenerative conditions linked to the accumulation in the brain of abnormal misfolded proteins. Such diseases include Prion diseases, Parkinson, Alzheimer, Amyotrophic lateral sclerosis (ALS), and many others. The contribution of the IRE1a pathway to the disease process, however, remains unknown. Our laboratory is particularly committed to investigate stress responses linked to irreversible ER damage and to understand how this pathway influences pathological conditions affecting the nervous system. Our research is focused on studying different aspects of IRE1a function and addresses two central questions:

• What are the molecular mechanism involved in the regulation of IRE1a by apoptosis-related proteins and
• What is the contribution of IREa pathway to pathological conditions affecting the nervous system.

Therefore, we are committed in first place to generate mechanistic insights about IRE1a signaling and, in second place, to apply this investigation to the study of neurodegenerative diseases linked to ER dysfunction.  Some of the diseases that we are currently investigating include Prion-related disorders, Huntington’s disease and Amyotrophic Lateral Sclerosis. In addition, we are currently developing therapeutic strategies to cure this disease using animal models and gene therapy strategies using interfering RNA and genetically modified viral vectors.
            In summary, we aim to enhance our understanding of the molecular mechanisms responsible for the adaptation to stress conditions affecting organelle function, and the means by which the UPR is interconnected with the main core apoptosis pathways. We believe that this investigation has particular relevance for the understanding of the cellular bases responsible for diseases in which protein misfolding is as a central cause, and that it may generate new clues about novel therapeutic strategies to treat these fatal diseases.

 

II RESEARCH
Three main areas:
1. APOPTOSIS, CELLULAR STRESS AND THE UNFOLDED PROTEIN RESPONSE

NON APOPOTIC ROLE OF DEATH PROTEINS

The viability of a cell strictly depends on the functional and structural integration of a number of subcellular organelles like the nucleus, mitochondria, lysosomes and the endoplasmic reticulum (ER).  Each organelle can sense stressful cellular conditions and initiate cellular responses either to adapt or to activate specific cell death signalling pathways promoting apoptosis if a critical threshold of damage has been reached. Deregulation of apoptosis contributes to a variety of human diseases, including autoimmunity, cancer, and neurodegeneration.

The induction of apoptosis ultimately converges upon the activation of cysteine proteases of the caspase family. The BCL-2 family proteins are located upstream at organelle membranes and control the activation of downstream caspases, representing a critical intracellular checkpoint proximal in the apoptotic pathway. The BCL-2 family is comprised of pro- and anti-apoptotic members (Fig. 1).  Genetic and biochemical analysis indicates that multidomain BAX and BAK function in concert as the essential gateway to the intrinsic cell death pathway operating at the mitochondria. The upstream BH3-only members respond to particular apoptotic signals and subsequently, either directly or indirectly, trigger the conformational activation of BAX and BAK, inducing their intramembranous homo-oligomerization and resultant permeabilization of the mitochondrial outer membrane.

Recent evidence indicates that members of all three subclasses of BCL-2 family proteins also localize to the ER membrane and have been shown to influence ER homeostasis, perhaps by modulating membrane permeability to calcium (Fig. 2).

 

 

 

 

 

 

 

 

The ER is the subcellular compartment in which proteins destined for the secretory pathway and lipids are synthesized. Any alterations in the ER function may cause ER stress and consequently cell death. The ER-stress response has been subdivided into two main components. First, an anti-apoptotic component is mediated by the upregulation of different chaperones and general decrease in protein synthesis (known as the unfolding protein response or UPR). If the stress level overcomes the ability of the cell to maintain ER homeostasis, the second component is activated, leading ultimately to the initiation of apoptosis.
Our laboratory recently described a new function for BCL-2 related proteins at the ER membrane where they control de activation of the UPR stress sensor IRE1a. IRE1a is a Ser/Thr protein kinase and endoribonuclease that, upon activation, initiates the unconventional splicing of the mRNA encoding the transcriptional factor X-Box-binding protein 1 (XBP-1) (Fig. 3).

 

In mammalian cells, a 26 nucleotide intron of xbp-1 mRNA is spliced out by activated IRE1a, leading to a shift in the coding reading frame. This splicing event promotes the expression of a more stable and potent transcriptional activator that controls the upregulation of a broad spectrum of UPR-related genes involved in protein folding, redox metabolism, ER-associated degradation and protein quality control (Fig. 3). Recently we showed that the pro-apoptotic proteins BAX and BAK regulate the UPR through a modulation of the  signaling. We are currently investigating the mechanism of amplitude of IRE1a this regulation and its relation to other apoptosis-related proteins (Fig. 4). Our general aim is to decipher the molecular connection between the UPR and the apoptosis machinery. This research will enhance our understanding of pathological conditions associated with irreversible organelle damage linked with many human diseases.

 

2. PROTEIN CONFORMATIONAL DISORDERS AND NEURODEGENERATION
Clinical Problem: The most common neurodegenerative diseases, such as Alzheimer’s Disease, Parkinson’s Disease, Amyotrophic lateral sclerosis (ALS), and Huntington’s disease, affect millions of people worldwide, but there is neither preventive nor curative therapy for them.  Elucidation of the genetic causes of the familial variants of these diseases has contributed much insight, but the pathogenesis of the far more common sporadic variants is unknown. Recent evidence suggests that perturbation of organelle homeostasis and endoplasmic reticulum (ER) stress correlates with disease onset and progression in animal models of ALS. Our goal is to determine whether the Unfolded Protein Response (UPR), an adaptive response against ER stress, contributes to the development of neuronal degeneration (Fig. 5). Overall, this project aims to increase our understanding of the molecular basis of protein conformational disorders and, use this knowledge to design possible new and effective therapeutic strategies to treat these fatal diseases.

APOPTOSIS AND ALS: ALS is a progressive adult-onset motoneuron disease characterized by muscle weakness, atrophy, paralysis and premature death.  The pathological hallmark of ALS is the selective degeneration of motoneurons in the spinal ventral horn, most brainstem nuclei and cerebral cortex. The majority of ALS patients lack a defined hereditary genetic component and are considered sporadic, while approximately 10% of cases are familial (FALS). Over 100 mutations in the gene encoding superoxide dismutase-1 (SOD1), which trigger the misfolding and abnormal aggregation of SOD1, have been genetically linked with FALS. Overexpression of human FALS-linked SOD1 mutations in transgenic mice provokes age-dependent protein aggregation, paralysis and motoneuron degeneration suggesting a direct involvement of misfolded SOD1 in the disease process. The primary mechanism by which mutations in SOD1 contribute to progressive motoneuron loss in FALS remains unknown. It has been proposed that motoneuron apoptosis is mediated by different mechanisms including mitochondrial dysfunction, altered axonal transport, endoplasmic reticulum (ER) stress and other non-neuronal components.  We have recently described the involvement of the BCL-2 pro-apoptotic gene BIM in the process of motoneuron loss in ALS in vivo. We are currently defining what is the contribution of the ER stress pathway to the disease process using transgenic animal and cellular models of the disease.

AUTOPHAGY AND ALS. Over 100 mutations in the gene encoding Superoxide Dismutase-1 (SOD1) are known to cause familial Amyotrophic lateral sclerosis (fALS), which triggers the misfolding and aggregation of SOD1. The primary mechanism responsible for the progressive motoneuron loss in ALS remains unknown. Recent evidence from different laboratories, however, implicates the participation of adaptive responses to stress at the endoplasmic reticulum (ER) in the disease process, a pathway known as the Unfolded Protein Response (UPR). In order to study the contribution of the ER stress pathway to fALS, we have targeted the expression of the transcription factor X-Box binding protein-1 (XBP-1) by creating a conditional knockout mouse. XBP-1 is the master regulator of the UPR and controls the expression of multiple proteins involved in protein folding and protein quality control. Unexpectedly, despite predictions that XBP-1 deficiency would enhance the severity of experimental ALS, we observed that these mice were more resistant to developing the disease, XBP-1-/- SOD1 transgenic mice displayed a significant increase in their life span accompanied by decreased motoneuron apoptosis. These effects were associated with increased autophagy in motoneurons, a cellular pathway involved in lysosome-mediated degradation of protein aggregates in other important neurological diseases. Similarly, in vitro experiments demonstrated that targeting XBP-1 with small interfering RNA in a motoneuron cell line drastically decreased the generation of toxic SOD1G93A and SOD1G85R protein aggregates due to enhanced autophagy, suggesting possible therapeutic benefits of targeting this pathway in ALS.
            In this project, we propose to further study the contribution of autophagy to fALS employing two independent approaches. First, our laboratory will establish and characterize the possible therapeutic benefits of increasing autophagy levels or decreasing ER stress signaling in animal models of fALS. Specifically, we intend to assess the effects of administrating the chemical chaperone TUDCA (known attenuator of ER stress) or rapamycin analogs (an activator of autophagy) on mutant SOD1 aggregation and neurotoxicity in a transgenic model of ALS. In addition, we plan to test the effects of oral administration of Trehalose, a new drug with chemical chaperone and autophagy enhancer activity. The drugs proposed have outstanding in vivo safety profiles and have been approved by the U.S. Food and Drug Administration for clinical use, and have proven efficiency in decreasing neurodegeneration in other mouse disease models. Secondly, in collaboration with Robert H. Brown (Massachusetts General Hospital) we plan to systematically measure the levels of ER stress and autophagy in spinal cord post-mortem samples from patients affected with sporadic and familial ALS. Overall, our long term objective is to increase our understanding of the molecular basis of ALS and, use this knowledge to design new therapeutic strategies to treat this fatal disease and other neuromuscular disorders.

General aim: Our general objective is to investigate the role of autophagy in the development of Amyotrophic lateral sclerosis. We also intend to define the possible therapeutic benefits of targeting the autophagy pathway using pharmacological approaches.

Specific Aim 1: (i) To assess the role of autophagy in mutant SOD1 pathogenesis in cellular models of fALS. (ii) To investigate the effects of chemical chaperones and autophagy activators in mutant SOD1 aggregation and toxicity in vitro.
Specific Aim 2. To test and compare the effects on the disease onset, motoneuron survival and SOD1 aggregation of treating pre-symptomatic and symptomatic mutant SOD1 transgenic mice with the chemical chaperone TUDCA, and the autophagy activators rapamycin and trehalose.
Specific Aim 3: To determine the levels of autophagy and ER stress markers in post-mortem samples of spinal cord from patients affected with sporadic and familial ALS.

 

ER STRESS AND PRION DISORDERS
Transmissible spongiform encephalopathies (TSEs), also known as prion disorders are a group of diseases that affect humans and animals and present a long incubation period.  Once the first clinical signs appear, the disease progression is relatively fast and death occurs in a short time.  Prion diseases are characterized by neurological dysfunction that may include dementia, ataxia and psychiatric disturbances.  The central molecular event in the pathogenesis of prion diseases is the conversion of the normal cellular prion protein, termed PrPC, into the pathological form denoted PrPSC (for scrapie associated PrP). In infectious forms of the disease, PrPSC formation from wild-type PrPC is initiated by the exposition to exogenous infectious agents, promoting a conformational transition from α-helical to β-sheet structure, resulting in the formation of PrPSC
Compelling evidence suggests the involvement of several signaling pathways in prion pathogenesis, including proteasome dysfunction, alterations in the protein maturation pathways and the unfolded protein response. We have reported that endoplasmic reticulum stress due to the PrP misfolding may be a critical factor mediating neuronal dysfunction in prion diseases in vivo in animal models of the disease and in human post-morten samples of patients affected with Creutzfeldt-Jacob disease, a human form of prion disorders. These findings have applications for developing novel strategies for treatment and early diagnosis of transmissible spongiform encephalopathies and other neurodegenerative diseases. We have recently described the involvement of the UPR in the misfolding and aggregation of pathological prion proteins. We are currently studying in more detail the basis of this regulation.

HUNTINGTON’S DISEASE AND THE UPR. 

Huntington disease (HD) is one of at least nine inherited neurodegenerative disorders caused by an extended polyglutamine (poly(Q)) stretch in the corresponding causative genes. Extended poly(Q) affecting the Huntingtin gene generates neurotoxic intracellular protein aggregates involved in the onset of progressive chorea, psychiatric symptoms, and dementia. Cellular studies imply the participation of adaptive responses to stress at the endoplasmic reticulum (ER) in the disease process, a pathway known as the Unfolded Protein Response (UPR).
            In order to study the contribution of the ER stress pathway to HD, we have targeted the expression of the three main UPR transcription factors X-Box binding protein-1 (XBP-1), Activating Transcription 4 and 6 (ATF4 and ATF6) with small interfering RNA. These transcription factors are the master regulators of the UPR and control the expression of multiple proteins involved in protein folding and quality control. As expected, targeting ATF4 and ATF6 increased mutant poly(Q)79 toxicity and aggregation in neuronal cell lines.  Unexpectedly, knocking-down XBP-1 drastically decreased the generation of toxic poly(Q) protein aggregates. These effects were associated with increased autophagy, a cellular pathway involved in the lysosome degradation of HD-intracellular inclusions. Our results suggest that each UPR signaling branch has distinct and opposing effects on mutant Htt pathogenesis. In this project, we propose to investigate and compare the contribution of ATF4 and XBP-1 to HD in vivo using knockout mouse models. Our laboratory recently described the generation of a brain specific knockout mice for XBP-1, which constitutes a unique tool to study the potential of targeting the pathway in the context of HD. Overall, this project aims to increase our understanding of the molecular basis of HD and use this knowledge to design new therapeutic strategies to treat this fatal disease.
           
Specific aims

Specific aim 1. Analyze the susceptibility of XBP-1 and ATF4 deficient primary neurons to the toxicity of mutant Htt.
Specific aim 2. Define and compare the contribution of XBP-1 and ATF4 to the generation of mutant Htt intracellular inclusions in vivo.
Specific aim 3. Assess the effects of XBP-1 and ATF4 deletion on striatal neuron loss induced by mutant Htt using a lentiviral base model.
Specific aim 4. Define the levels of ER stress markers on a HD transgenic mouse model at different stages of the disease using a lentiviral base model.
Specific aim 5. Address the susceptibility of XBP-1 deficient mice to develop HD using transgenic mouse models.

XBP-1 AND PARKINSON'S DISEASE
Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting at least 1% of the population over 55 years old. The major clinical symptom of PD is impairment of motor control as a result from extensive dopaminergic neuron death in the substantia nigra pars compacta (SNpc). One of the pathological hallmarks of the disorder is the presence of Lewy bodies (LBs) inclusions, which contain aggregated and misfolded α-synuclein protein. The mechanism involved in dopaminergic neuron loss in PD remains speculative. Recent evidence, however, implicates participation of adaptive responses to stress at the endoplasmic reticulum (ER) in the disease process, a pathway known as the Unfolded Protein Response (UPR). A recent study demonstrated that the earliest defect following α-synuclein expression is a block in ER to Golgi vesicular trafficking with concomitant UPR activation. More importantly, activation of the UPR was recently described in post-mortem tissue from sporadic PD human cases. Toxicological models resembling sporadic PD also suggest an involvement of ER stress in the pathology. The contribution of the UPR to PD has never been addressed directly, and genetic manipulation of the UPR is required to define the functional role of this stress pathway to PD.
We have obtained data from transgenic mice expressing human mutant alpha-synucleinA53T, observing upregulation of several ER stress markers in the brain of pre-symptomatic and symptomatic mice. Similarly, a pharmacological model of PD using 6-hydroxydopamine (6-OHDA) triggers the activation of XBP-1, associated with the processing of its mRNA. Our laboratory intend to analyze the role of XBP-1 in PD using both genetic and pharmacological models. Overall, our long term objective is to increase the molecular understanding of PD, using this knowledge to design new therapeutic strategies to treat this fatal disease.

General aim: Our general objective is to investigate the role of the Unfolded Protein Response (UPR) in the development of Parkinson's disease. In the long term, we also intend to define the possible therapeutic benefits of alleviating stress on the ER using pharmacological approaches in a disease context.

Specific aim 1: To assess and compare the role of XBP-1 in the toxicity of mutant a-synuclein and 6-OHDA in cellular models of PD.
Specific aim 2: To define the role of XBP-1 in PD in vivo using genetic and sporadic model of the diseases.

3. THERAPEUTIC STRATEGIES TO REDUCE BRAIN DAMAGE AND NEURONAL DYSFUNCTION: Three approaches, Small interfering RNA, lentiviral vectors and small chemical molecules.
Defective handling of proteins is a central feature of major neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease. The discovery that neuronal dysfunction or degeneration can be caused by mutations in single cellular proteins has provided new opportunities to model the underlying disease processes by genetic modification of cells in vitro or by generation of transgenic animals carrying the disease causing gene. Recent developments in recombinant viral-vector technology have opened up an interesting alternative possibility, based on direct gene transfer to selected subsets of neurons in the brain. Using highly efficient lentivirus vectors, recent reports have shown that therapeutic genes can be delivered in vivo to the targeted tissue. The advantages of these vectors are well established and include their stable chromosomal insertion (allowing the continue delivery of the therapeutic gene), low immunogenicity (decreasing the side effects) and high tropism (increasing the range of biomedical applications of target tissues).
In recent years, an alternative approach to inactivate disease causing genes has been developed that is highly specific and safer than the use of chemical inhibitors. Mammalian cells can be transduced with lentiviruses carrying expression cassettes that encode short hairpin RNAs (shRNAs) to generate gene specific interfering RNAs that induce specific degradation of the target mRNA. This approach results in stable and highly effective gene suppression in a variety of mammalian cell types in vivo and in vitro.

 

 

 

 

 

 

 

 

 

 

We are currently employing lentiviral vectors to deliver shRNA in vivo as a therapeutic tool for gene therapy (Fig. 6). We are employing the shRNA strategy to inactivate many different components of stress responses and thereby identify common determinants of neuronal apoptosis in diseases such as Huntington, Prion Disorders and Amyotrophic Lateral Sclerosis. Currently experimental strategies are being developed to deliver shRNA-containing lentivirus to the affected brain areas by stereotaxis injections in transgenic animal models of the mentioned diseases. These experiments seek to define the possible beneficial effects of targeting specific signaling pathways in a context relevant to important human diseases. The other unique advantage of using lentivirus and shRNA is the inactivation of a gene of interest in a living animal as an alternative model to the knockout mice.
In collaboration with Bruno Antonsson at the Biotechnological company SERONO in Switzerland, we have developed small molecules to block apoptosis by targeting the pro-apoptotic gene BAX.  We proved a beneficial effect of these drugs in animal models of brain ischemia. We are currently characterizing compound that can reduce the stress at the ER by stabilizing protein folding. These prototypic drugs, called “chemical chaperones”, will be employed in our disease models of neurodegeneration to address the therapeutic effects of targeting the pathway.

Other research lines
Analysis of the impact of Air Pollution in Santiago in health using animal models.
Concern over possible health effects of environmental particulate matter ≤2.5 μm (PM2.5) [U.S. Environmental Protection Agency (EPA) 2004] has stimulated numerous studies of its chemical/physical properties, the sources that contribute the most hazardous components, and biological mechanisms for the adverse effects. Although epidemiologic
studies indicate that significant effects are often associated with PM2.5 exposure, the magnitude of the effect varies with location. However, few studies have directly compared the effects of ambient respirable PM from different locations in vivo. Such studies are critical to rational regulation of PM based on source/composition/toxicity relationships rather than size alone. In our the present studies with the MARIO MOLINA CENTER CHILE we are using intratracheal instillation of Santiago air samples to compare toxicity of PM2.5 collected during summer or winter and different areas of Santiago from four with different contributing sources. We are characterizing and monitoring the levels of lung damage and dysfunction in animal models as a measure of the impact on health of the air in the City.
This work is lead by the Mario Molina Center Chile and funded by the government agency CONAMA. www.cmmolina.cl