Gabriela Mercado

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.

An ERcentric view of Parkinson's disease.

Parkinsons disease (PD) is the second most common neurodegenerative disease and is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta and the accumulation of intracellular inclusions containing α-synuclein (αSyn). Growing evidence from studies in human PD brain, in addition to genetic and toxicological models, indicates that endoplasmic reticulum (ER) stress is a common feature of the disease and contributes to neurodegeneration. Recent reports place ER dysfunction as an early component of PD pathogenesis, and in this article we review the impact of ER stress in PD models and discuss the multiple mechanisms underlying the perturbation of secretory pathway function. Possible therapeutic strategies to mitigate ER stress in the context of PD are also discussed.

Control of dopaminergic neuron survival by the unfolded protein response transcription factor XBP1

Significance The selective loss of dopaminergic neurons is characteristic of Parkinson disease (PD). Protein folding stress is a salient feature of PD. This study uncovers a previously undefined function of a major unfolded protein response (UPR) transcription factor (XBP1) in supporting the survival of nigral dopaminergic neurons at basal levels and under pathological conditions. Our results reveal an important role for a canonical UPR pathway in the maintenance of dopaminergic neuron proteostasis, which also could be relevant to understand the selective neuronal vulnerability observed in Parkinson disease. Parkinson disease (PD) is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta (SNpc). Although growing evidence indicates that endoplasmic reticulum (ER) stress is a hallmark of PD, its exact contribution to the disease process is not well understood. Here we report that developmental ablation of X-Box binding protein 1 (XBP1) in the nervous system, a key regulator of the unfolded protein response (UPR), protects dopaminergic neurons against a PD-inducing neurotoxin. This survival effect was associated with a preconditioning condition that resulted from induction of an adaptive ER stress response in dopaminergic neurons of the SNpc, but not in other brain regions. In contrast, silencing XBP1 in adult animals triggered chronic ER stress and dopaminergic neuron degeneration. Supporting this finding, gene therapy to deliver an active form of XBP1 provided neuroprotection and reduced striatal denervation in animals injected with 6-hydroxydopamine. Our results reveal a physiological role of the UPR in the maintenance of protein homeostasis in dopaminergic neurons that may help explain the differential neuronal vulnerability observed in PD.

ER stress and Parkinson's disease: Pathological inputs that converge into the secretory pathway.

The major clinical feature of Parkinsons disease (PD) is impairment in motor control as a result of extensive dopaminergic neuron loss in the substantia nigra pars compacta. The central pathological hallmark of PD is the formation of neuronal cytoplasmic inclusions of insoluble proteins called Lewy bodies, of which fibrillar aggregates of misfolded αSynuclein are the major components. Despite intense research on the pathogenic mechanism that trigger neuronal loss and disease progression, the neurogenesis of PD remains unknown. However, studies on genetics of PD have identified specific genes and proteins linked to this disease. Genetic mutations linked with different forms of familial PD have unveiled a closer relationship between pathology and impairments at different points in the secretory pathway. Accumulation of misfolded/unfolded proteins in the endoplasmic reticulum and disruptions in protein clearance mechanisms result in activation of an adaptive stress pathway known as the unfolded protein response (UPR). UPR signaling is mediated by three stress sensors that induce independent and convergent signaling branches that help to maintain homeostasis, or eventually trigger cell death under chronic stress conditions. Signs of ER stress are observed in post-mortem tissue from sporadic human PD cases and in most animal models of the disease, implicating all three branches of this cellular response. However, the exact contribution of the UPR in the progression of PD or in dopaminergic neuron survival is not yet well understood. A large number of studies reveal a clear activation of the UPR in toxicological models resembling sporadic PD, where ATF6, XBP1 and CHOP have a functional role in controlling dopaminergic neuron survival in neurotoxin-based models of PD in vivo. Also pharmacological and gene therapy approaches aimed to target different points of this pathway have revealed an important functional role in PD pathogenesis. This article is part of a Special Issue entitled SI:ER stress.

Functional Role of the Disulfide Isomerase ERp57 in Axonal Regeneration.

ERp57 (also known as grp58 and PDIA3) is a protein disulfide isomerase that catalyzes disulfide bonds formation of glycoproteins as part of the calnexin and calreticulin cycle. ERp57 is markedly upregulated in most common neurodegenerative diseases downstream of the endoplasmic reticulum (ER) stress response. Despite accumulating correlative evidence supporting a neuroprotective role of ERp57, the contribution of this foldase to the physiology of the nervous system remains unknown. Here we developed a transgenic mouse model that overexpresses ERp57 in the nervous system under the control of the prion promoter. We analyzed the susceptibility of ERp57 transgenic mice to undergo neurodegeneration. Unexpectedly, ERp57 overexpression did not affect dopaminergic neuron loss and striatal denervation after injection of a Parkinson’s disease-inducing neurotoxin. In sharp contrast, ERp57 transgenic animals presented enhanced locomotor recovery after mechanical injury to the sciatic nerve. These protective effects were associated with enhanced myelin removal, macrophage infiltration and axonal regeneration. Our results suggest that ERp57 specifically contributes to peripheral nerve regeneration, whereas its activity is dispensable for the survival of a specific neuronal population of the central nervous system. These results demonstrate for the first time a functional role of a component of the ER proteostasis network in peripheral nerve regeneration.

ER proteostasis disturbances in Parkinson's disease: novel insights

Parkinsons disease (PD) is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta (SNpc). Proteostasis impairment at the level of the endoplasmic reticulum (ER) is emerging as a driving factor of dopaminergic neuron loss in PD. ER stress engages the activation of an adaptive reaction known as the unfolded protein response (UPR) to recover proteostasis or trigger apoptosis of damaged cells. The therapeutic potential of the UPR as a target has been recently validated using pharmacological and gene therapy approaches. A complex view is emerging where ER stress may have a dual role in PD, both in maintaining cell survival during initial stages of the diseases and trigger neuronal degeneration when the stress levels are sustained. Here we overview recent advances in determining the impact of ER stress to PD. PD is a progressive neurodegenerative disease that affects movement control, characterized by the loss of dopaminergic neurons in the SNpc. In most PD cases the presence of intracellular inclusions, termed Lewy bodies (LBs) is observed, where fibrillar aggregates of αSynuclein constitute a major component. Many cellular processes are altered in PD, including redox control, mitochondrial function, autophagy/lysosomal function, protein quality control mechanisms, and vesicle trafficking, among other processes. Accumulating evidence supports disruption in the secretory pathway as a triggering factor of proteostasis dysfunction in PD, mediating in part the selective degeneration of dopaminergic neurons (Chua and Tang, 2013; Mercado et al., 2013). Importantly, in addition to PD, ER stress is emerging as a relevant driver of most common neurodegenerative diseases (Hetz and Mollereau, 2014). ER stress activates the UPR, a complex signaling transduction pathway that mediates cellular adaptation to restore ER function (reviewed in Ron and Walter, 2007; Hetz, 2012). In this article we discuss recent insights on the significance of ER stress as a driver of dopaminergic neuron loss in PD and the potential of targeting UPR components to augment the homeostatic capacity of the ER and reduce pro-apoptotic signals.

Structural characterization of microcin E492 amyloid formation: Identification of the precursors.

Microcin E492 is a low-molecular weight, channel-forming bacteriotoxin that generates amyloid structures. Using electron microscopy and image processing techniques several structural conformations can be observed. Prior to the conditions that induce amyloid formation and at its initial stage, microcin E492 molecules can be found in two main types of oligomers: a pentameric, pore-like structure consisting of globular monomers of ∼25Å diameter, and long filaments made up of stacked pentamers. The equilibrium between these structures depends on the properties of the solvent, because samples kept in methanol mainly show the pentameric structure. Amyloid induction in aqueous solvent reveals the presence, together with the above mentioned structures, of several amyloid structures such as flat and helical filaments. In addition, X-ray diffraction analysis demonstrated that the fibrils formed by microcin E492 presented cross-β structure, a distinctive property of amyloid fibrils. Based on the study of the observed structures we propose that microcin E492 has two conformations: a native one that assembles mainly into a pentameric structure, which functions as a pore, and an amyloid conformation which results in the formation of different types of amyloid filaments.

The Production In Vivo of Microcin E492 with Antibacterial Activity Depends on Salmochelin and EntF

Microcin E492 is a channel-forming bacteriocin that is found in two forms, namely, a posttranslationally modified form obtained by the covalent linkage of salmochelin-like molecules to serine 84 and an unmodified form. The production of modified microcin E492 requires the synthesis of enterochelin, which is subsequently glycosylated by MceC and converted into salmochelin. mceC mutants produced inactive microcin E492, and this phenotype was reversed either by complementation with iroB from Salmonella enterica or by the addition of exogenous salmochelin. Cyclic salmochelin uptake by Escherichia coli occurred mainly through the outer membrane catecholate siderophore receptor Fiu. The production of inactive microcin E492 by mutants in entB and entC was reverted by the addition of the end product of the respective mutated pathway (2,3-dihydroxybenzoic acid and enterochelin/salmochelin, respectively), while mutants in entF did not produce active microcin E492 in the presence of enterochelin or salmochelin. The EntF adenylation domain was the only domain required for this microcin E492 maturation step. Inactivation of the enzymatic activity of this domain by site-directed mutagenesis did not prevent the synthesis of active microcin E492 in the presence of salmochelin, indicating that the adenylation activity is not essential for the function of EntF at this stage of microcin E492 maturation.

Microcin E492 Amyloid Formation Is Retarded by Posttranslational Modification

Microcin E492, a channel-forming bacteriocin with the ability to form amyloid fibers, is exported as a mixture of two forms: unmodified (inactive) and posttranslationally modified at the C terminus with a salmochelin-like molecule, which is an essential modification for conferring antibacterial activity. During the stationary phase, the unmodified form accumulates because expression of the maturation genes mceIJ is turned off, and microcin E492 is rapidly inactivated. The aim of this work was to demonstrate that the increase in the proportion of unmodified microcin E492 augments the ability of this bacteriocin to form amyloid fibers, which in turn decreases antibacterial activity. To this end, strains with altered proportions of the two forms were constructed. The increase in the expression of the maturation genes augmented the antibacterial activity during all growth phases and delayed the loss of activity in the stationary phase, while the ability to form amyloid fibers was markedly reduced. Conversely, a higher expression of microcin E492 protein produced concomitant decreases in the levels of the modified form and in antibacterial activity and a substantial increase in the ability to form amyloid fibers. The same morphology for these fibers, including those formed by only the unmodified version, was observed. Moreover, seeds formed using exclusively the nonmodified form were remarkably more efficient in amyloid formation with a shorter lag phase, indicating that the nucleation process is probably improved. Unmodified microcin E492 incorporation into amyloid fibers was kinetically more efficient than the modified form, probably due to the existence of a conformation that favors this process.

Targeting PERK signaling with the small molecule GSK2606414 prevents neurodegeneration in a model of Parkinson's disease

Parkinsons disease (PD) is the second most common neurodegenerative disorder, leading to the progressive decline of motor control due to the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Accumulating evidence suggest that altered proteostasis is a salient feature of PD, highlighting perturbations to the endoplasmic reticulum (ER), the main compartment involved in protein folding and secretion. PERK is a central ER stress sensor that enforces adaptive programs to recover homeostasis through a block of protein translation and the induction of the transcription factor ATF4. In addition, chronic PERK signaling results in apoptosis induction and neuronal dysfunction due to the repression in the translation of synaptic proteins. Here we confirmed the activation of PERK signaling in postmortem brain tissue derived from PD patients and three different rodent models of the disease. Pharmacological targeting of PERK by the oral administration of GSK2606414 demonstrated efficient inhibition of the pathway in the SNpc after experimental ER stress stimulation. GSK2606414 protected nigral-dopaminergic neurons against a PD-inducing neurotoxin, improving motor performance. The neuroprotective effects of PERK inhibition were accompanied by an increase in dopamine levels and the expression of synaptic proteins. However, GSK2606414 treated animals developed secondary effects possibly related to pancreatic toxicity. This study suggests that strategies to attenuate ER stress levels may be effective to reduce neurodegeneration in PD.