Vicente Valenzuela

Trehalose delays the progression of amyotrophic lateral sclerosis by enhancing autophagy in motoneurons

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuron disease with no current effective treatment. Accumulation of abnormal protein inclusions containing SOD1, TARDBP, FUS, among other proteins, is a pathological hallmark of ALS. Autophagy is the major degradation pathway involved in the clearance of damaged organelles and protein aggregates. Although autophagy has been shown to efficiently degrade ALS-linked mutant protein in cell culture models, several studies suggest that autophagy impairment may also contribute to disease pathogenesis. In this report, we tested the potential use of trehalose, a disaccharide that induces MTOR-independent autophagy, in the development of experimental ALS. Administration of trehalose to mutant SOD1 transgenic mice significantly prolonged life span and attenuated the progression of disease signs. These effects were associated with decreased accumulation of SOD1 aggregates and enhanced motoneuron survival. The protective effects of trehalose were associated with increased autophagy levels in motoneurons. Cell culture experiments demonstrated that trehalose led to mutant SOD1 degradation by autophagy in NSC34 motoneuron cells and also protected primary motoneurons against the toxicity of conditioned media from mutant SOD1 transgenic astrocytes. At the mechanistic level, trehalose treatment led to a significant upregulation in the expression of key autophagy-related genes at the mRNA level including Lc3, Becn1, Sqstm1 and Atg5. Consistent with these changes, trehalose administration enhanced the nuclear translocation of FOXO1, an important transcription factor involved in the activation of autophagy in neurons. This study suggests a potential use of trehalose and enhancers of MTOR-independent autophagy for the treatment of ALS.

Activation of the unfolded protein response enhances motor recovery after spinal cord injury

Spinal cord injury (SCI) is a major cause of paralysis, and involves multiple cellular and tissular responses including demyelination, inflammation, cell death and axonal degeneration. Recent evidence suggests that perturbation on the homeostasis of the endoplasmic reticulum (ER) is observed in different SCI models; however, the functional contribution of this pathway to this pathology is not known. Here we demonstrate that SCI triggers a fast ER stress reaction (1–3 h) involving the upregulation of key components of the unfolded protein response (UPR), a process that propagates through the spinal cord. Ablation of X-box-binding protein 1 (XBP1) or activating transcription factor 4 (ATF4) expression, two major UPR transcription factors, leads to a reduced locomotor recovery after experimental SCI. The effects of UPR inactivation were associated with a significant increase in the number of damaged axons and reduced amount of oligodendrocytes surrounding the injury zone. In addition, altered microglial activation and pro-inflammatory cytokine expression were observed in ATF4 deficient mice after SCI. Local expression of active XBP1 into the spinal cord using adeno-associated viruses enhanced locomotor recovery after SCI, and was associated with an increased number of oligodendrocytes. Altogether, our results demonstrate a functional role of the UPR in SCI, offering novel therapeutic targets to treat this invalidating condition.Cell Death and Disease (2012) 3, e272; doi:10.1038/cddis.2012.8; published online 16 February 2012

Pathogenic role of BECN1/Beclin 1 in the development of amyotrophic lateral sclerosis

Pharmacological activation of autophagy is becoming an attractive strategy to induce the selective degradation of aggregate-prone proteins. Recent evidence also suggests that autophagy impairment may underlie the pathogenesis of several neurodegenerative diseases. Mutations in the gene encoding SOD1 (superoxide disumutase 1) trigger familial amyotrophic lateral sclerosis (ALS), inducing its misfolding and aggregation and the progressive loss of motoneurons. It is still under debate whether autophagy has a protective or detrimental role in ALS. Here we evaluate the impact of BECN1/Beclin 1, an essential autophagy regulator, in ALS. BECN1 levels were upregulated in both cells and animals expressing mutant SOD1. To evaluate the impact of BECN1 to the pathogenesis of ALS in vivo, we generated mutant SOD1 transgenic mice heterozygous for Becn1. We observed an unexpected increase in life span of mutant SOD1 transgenic mice haploinsufficient for Becn1 compared with littermate control animals. These effects were accompanied by enhanced accumulation of SQSTM1/p62 and reduced levels of LC3-II, and an altered equilibrium between monomeric and oligomeric mutant SOD1 species in the spinal cord. At the molecular level, we detected an abnormal interaction of mutant SOD1 with the BECN1-BCL2L1 complex that may impact autophagy stimulation. Our data support a dual role of BECN1 in ALS and depict a complex scenario in terms of predicting the effects of manipulating autophagy in a disease context.

Measurement of autophagy flux in the nervous system in vivo.

Accurate methods to measure autophagic activity in vivo in neurons are not available, and most of the studies are based on correlative and static measurements of autophagy markers, leading to conflicting interpretations. Autophagy is an essential homeostatic process involved in the degradation of diverse cellular components including organelles and protein aggregates. Autophagy impairment is emerging as a relevant factor driving neurodegeneration in many diseases. Moreover, strategies to modulate autophagy have been shown to provide protection against neurodegeneration. Here we describe a novel and simple strategy to express an autophagy flux reporter in the nervous system of adult animals by the intraventricular delivery of adeno-associated viruses (AAV) into newborn mice. Using this approach we efficiently expressed a monomeric tandem mCherry-GFP-LC3 construct in neurons of the peripheral and central nervous system, allowing the measurement of autophagy activity in pharmacological and disease settings.

ER Dysfunction and Protein Folding Stress in ALS

Amyotrophic lateral sclerosis (ALS) is the most frequent paralytic disease in adults. Most ALS cases are considered sporadic with no clear genetic component. The disruption of protein homeostasis due to chronic stress responses at the endoplasmic reticulum (ER) and the accumulation of abnormal protein inclusions are extensively described in ALS mouse models and patient-derived tissue. Recent studies using pharmacological and genetic manipulation of the unfolded protein response (UPR), an adaptive reaction against ER stress, have demonstrated a complex involvement of the pathway in experimental models of ALS. In addition, quantitative changes in ER stress-responsive chaperones in body fluids have been proposed as possible biomarkers to monitor the disease progression. Here we review most recent advances attributing a causal role of ER stress in ALS.

Functional Contribution of the Transcription Factor ATF4 to the Pathogenesis of Amyotrophic Lateral Sclerosis

Endoplasmic reticulum (ER) stress represents an early pathological event in amyotrophic lateral sclerosis (ALS). ATF4 is a key ER stress transcription factor that plays a role in both adaptation to stress and the activation of apoptosis. Here we investigated the contribution of ATF4 to ALS. ATF4 deficiency reduced the rate of birth of SOD1G86R transgenic mice. The fraction of ATF4−/−-SOD1G85R transgenic mice that were born are more resistant to develop ALS, leading to delayed disease onset and prolonged life span. ATF4 deficiency completely attenuated the induction of pro-apoptotic genes, including BIM and CHOP, and also led to quantitative changes in the ER protein homeostasis network. Unexpectedly, ATF4 deficiency enhanced mutant SOD1 aggregation at the end stage of the disease. Studies in the motoneuron cell line NSC34 demonstrated that knocking down ATF4 enhances mutant SOD1 aggregation possibly due to alteration in the redox status of the cell. Our results support a functional role of ATF4 in ALS, offering a novel target for disease intervention.

ALS-linked protein disulfide isomerase variants cause motor dysfunction

Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) are ER foldases identified as possible ALS biomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized four ALS‐linked mutations recently identified in two major PDI genes, PDIA1 and PDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of these PDI variants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutant PDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of these PDI mutants. Finally, targeting ERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifies ER proteostasis imbalance as a risk factor for ALS, driving initial stages of the disease.

Gene therapy to target ER stress in brain diseases.

Gene therapy based on the use of Adeno-associated viruses (AAVs) is emerging as a safe and stable strategy to target molecular pathways involved in a variety of brain diseases. Endoplasmic reticulum (ER) stress is proposed as a transversal feature of most animal models and clinical samples from patients affected with neurodegenerative diseases. Manipulation of the unfolded protein response (UPR), a major homeostatic reaction under ER stress conditions, had proved beneficial in diverse models of neurodegeneration. Although increasing number of drugs are available to target ER stress, the use of small molecules to treat chronic brain diseases is challenging because of poor blood brain barrier permeability and undesirable side effects due to the role of the UPR in the physiology of peripheral organs. Gene therapy is currently considered a possible future alternative to circumvent these problems by the delivery of therapeutic agents to selective regions and cell types of the nervous system. Here we discuss current efforts to design gene therapy strategies to alleviate ER stress on a disease context. This article is part of a Special Issue entitled SI:ER stress.

A new method to measure autophagy flux in the nervous system.

A current need in the neuroscience field is a simple method to monitor autophagic activity in vivo in neurons. Until very recently, most reports have been based on correlative and static determinations of the expression levels of autophagy markers in the brain, generating conflicting interpretations. Autophagy is a fundamental process mediating the degradation of diverse cellular components, including organelles and protein aggregates at basal levels, whereas alterations in the process (i.e., autophagy impairment) operate as a pathological mechanism driving neurodegeneration in most prevalent diseases. We have recently described a new simple method to deliver and express an autophagy flux reporter through the peripheral and central nervous system of mice by the intracerebroventricular delivery of adeno-associated viruses (AAV) into newborn mice. We obtained a wide expression of a monomeric tandem mCherry-GFP-LC3 construct in neurons through the nervous system and demonstrated efficient and accurate measurements of LC3 flux after pharmacological stimulation of the pathway or in disease settings of axonal damage. Here we discuss the possible applications of this new method to assess autophagy activity in neurons in vivo.

Injury to the nervous system: A look into the ER.

Injury to the central or peripheral nervous systems leads to the loss of cognitive and/or sensorimotor capabilities that still lack an effective treatment. Although injury to the nervous system involves multiple and complex molecular factors, alteration to protein homeostasis is emerging as a relevant pathological mechanism. In particular, chronic endoplasmic reticulum (ER) stress is proposed as a possible driver of neuronal dysfunction in conditions such as spinal cord injury, stroke and damage to peripheral nerves. Importantly, manipulation of the unfolded protein response (UPR), a homeostatic pathway engaged by ER stress, has proved effective in improving cognitive and motor recovery after nervous system injury. Here we provide an overview on recent findings depicting a functional role of the UPR to the functional recovery after injury in the peripheral and central nervous systems. This article is part of a Special Issue entitled SI:ER stress.