Tauroursodeoxycholic bile acid arrests axonal degeneration by inhibiting the unfolded protein response in X-linked adrenoleukodystrophy.

The activation of the highly conserved unfolded protein response (UPR) is prominent in the pathogenesis of the most prevalent neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS), which are classically characterized by an accumulation of aggregated or misfolded proteins. This activation is orchestrated by three endoplasmic reticulum (ER) stress sensors: PERK, ATF6 and IRE1. These sensors transduce signals that induce the expression of the UPR gene programme. Here, we first identified an early activator of the UPR and investigated the role of a chronically activated UPR in the pathogenesis of X-linked adrenoleukodystrophy (X-ALD), a neurometabolic disorder that is caused by ABCD1 malfunction; ABCD1 transports very long-chain fatty acids (VLCFA) into peroxisomes. The disease manifests as inflammatory demyelination in the brain or and/or degeneration of corticospinal tracts, thereby resulting in spastic paraplegia, with the accumulation of intracellular VLCFA instead of protein aggregates. Using X-ALD mouse model (Abcd1 - and Abcd1 - /Abcd2 -/- mice) and X-ALD patient's fibroblasts and brain samples, we discovered an early engagement of the UPR. The response was characterized by the activation of the PERK and ATF6 pathways, but not the IRE1 pathway, showing a difference from the models of AD, PD or ALS. Inhibition of PERK leads to the disruption of homeostasis and increased apoptosis during ER stress induced in X-ALD fibroblasts. Redox imbalance appears to be the mechanism that initiates ER stress in X-ALD. Most importantly, we demonstrated that the bile acid tauroursodeoxycholate (TUDCA) abolishes UPR activation, which results in improvement of axonal degeneration and its associated locomotor impairment in Abcd1 - /Abcd2 -/- mice. Altogether, our preclinical data provide evidence for establishing the UPR as a key drug target in the pathogenesis cascade. Our study also highlights the potential role of TUDCA as a treatment for X-ALD and other axonopathies in which similar molecular mediators are implicated.

Functional genomic analysis unravels a metabolic-inflammatory interplay in adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is an inherited disorder characterized by axonopathy and demyelination in the central nervous system and adrenal insufficiency. Main X-ALD phenotypes are: (i) an adult adrenomyeloneuropathy (AMN) with axonopathy in spinal cords, (ii) cerebral AMN with brain demyelination (cAMN) and (iii) a childhood variant, cALD, characterized by severe cerebral demyelination. Loss of function of the ABCD1 peroxisomal fatty acid transporter and subsequent accumulation of very-long-chain fatty acids (VLCFAs) are the common culprits to all forms of X-ALD, an aberrant microglial activation accounts for the cerebral forms, whereas inflammation allegedly plays no role in AMN. How VLCFA accumulation leads to neurodegeneration and what factors account for the dissimilar clinical outcomes and prognosis of X-ALD variants remain elusive. To gain insights into these questions, we undertook a transcriptomic approach followed by a functional-enrichment analysis in spinal cords of the animal model of AMN, the Abcd1(-) null mice, and in normal-appearing white matter of cAMN and cALD patients. We report that the mouse model shares with cAMN and cALD a common signature comprising dysregulation of oxidative phosphorylation, adipocytokine and insulin signaling pathways, and protein synthesis. Functional validation by quantitative polymerase chain reaction, western blots and assays in spinal cord organotypic cultures confirmed the interplay of these pathways through IkB kinase, being VLCFA in excess a causal, upstream trigger promoting the altered signature. We conclude that X-ALD is, in all its variants, a metabolic/inflammatory syndrome, which may offer new targets in X-ALD therapeutics.

ß-Catenin determines upper airway progenitor cell fate and preinvasive squamous lung cancer progression by modulating epithelial-mesenchymal transition.

Human lung cancers, including squamous cell carcinoma (SCC) are a leading cause of death and, whilst evidence suggests that basal stem cells drive SCC initiation and progression, the mechanisms regulating these processes remain unknown. In this study we show that ß-catenin signalling regulates basal progenitor cell fate and subsequent SCC progression. In a cohort of preinvasive SCCs we established that elevated basal cell ß-catenin signalling is positively associated with increased disease severity, epithelial proliferation and reduced intercellular adhesiveness. We demonstrate that transgene-mediated ß-catenin inhibition within keratin 14-expressing basal cells delayed normal airway repair while basal cell-specific ß-catenin activation increased cell proliferation, directed differentiation and promoted elements of early epithelial-mesenchymal transition (EMT), including increased Snail transcription and reduced E-cadherin expression. These observations are recapitulated in normal human bronchial epithelial cells in vitro following both pharmacological ß-catenin activation and E-cadherin inhibition, and mirrored our findings in preinvasive SCCs. Overall, the data show that airway basal cell ß-catenin determines cell fate and its mis-expression is associated with the development of human lung cancer.

Cell stress induces TDP-43 pathological changes associated with ERK1/2 dysfunction: implications in ALS.

TDP-43 has been implicated in the pathogenesis of amyotrophic lateral sclerosis and other neurodegenerative diseases. Here we demonstrate, using neuronal and spinal cord organotypic culture models, that chronic excitotoxicity, oxidative stress, proteasome dysfunction and endoplasmic reticulum stress mechanistically induce mislocalization, phosphorylation and aggregation of TDP-43. This is compatible with a lack of function of this protein in the nucleus, specially in motor neurons. The relationship between cell stress and pathological changes of TDP-43 also includes a dysfunction in the survival pathway mediated by mitogen-activated protein kinase/extracellular signal-regulated kinases (ERK1/2). Thus, under stress conditions, neurons and other spinal cord cells showed cytosolic aggregates containing ERK1/2. Moreover, aggregates of abnormal phosphorylated ERK1/2 were also found in the spinal cord in amyotrophic lateral sclerosis (ALS), specifically in motor neurons with abnormal immunoreactive aggregates of phosphorylated TDP-43. These results demonstrate that cellular stressors are key factors in neurodegeneration associated with TDP-43 and disclose the identity of ERK1/2 as novel players in the pathogenesis of ALS.

Cell death and learning impairment in mice caused by in vitro modified pro-NGF can be related to its increased oxidative modifications in Alzheimer disease.

Pro-nerve growth factor (pro-NGF) is expressed at increased levels in Alzheimer's disease (AD)-affected brains and is able to induce cell death in cultures; however, the reasons for these phenomena remain elusive. Here we show that pro-NGF in human AD-affected hippocampus and entorhinal cortex is modified by advanced glycation and lipoxidation end-products in a stage-dependent manner. These modifications block pro-NGF processing to mature NGF, thus making the proneurotrophin especially effective in inducing apoptosis of PC12 cells in culture through the p75 neurotrophin receptor. The processing of advanced glycation and lipoxidation end-products in vitro modified recombinant human pro-NGF is severely impaired, as evidenced by Western blot and by examining its physiological functionality in cell cultures. We also report that modified recombinant human pro-NGF, as well as pro-NGF isolated from human brain affected by AD, cause impairment of learning tasks when administered intracerebroventricularly in mice, which correlates with AD-associated learning impairment. Taken together, the data we present here offer a novel pathway of ethiopathogenesis in AD caused by advanced glycation and lipoxidation end-products modification of pro-NGF.

Increased oxidation, glycoxidation, and lipoxidation of brain proteins in prion disease.

The basic molecular underpinnings of the pathological changes that unfold in prion disease remain elusive. A key role of increased oxidative stress has been hypothesized. Given the transient nature of most intermediate molecules implicated, increased oxidative stress is better assessed by quantitating the damage it causes to macromolecules. We used mass spectrometry-based methods to measure specific products of protein oxidation, glycoxidation, and lipoxidation in brains from patients suffering from Creutzfeldt-Jakob disease and Syrian hamsters affected by scrapie. In both cases, increased amounts of glutamic and aminoadipic semialdehydes, products of metal-catalyzed oxidation, malondialdehydelysine (a product of lipoxidation), N-epsilon-carboxyethyllysine (a product of glycoxidation), and N-epsilon-carboxymethyllysine (generated by lipoxidation and glycoxidation) were measured. PrP(Sc), the infectious isoform of the prion protein that accumulates in prion disease, was itself shown to be a target of increased oxidative modification. These changes were accompanied by alterations in fatty acid composition and increased phosphorylation of ERK(1/2) and p38, protein kinases known to respond to increased flows of ROS. These data support an important role of oxidative damage in the pathology of prion disease.

Maillard reaction versus other nonenzymatic modifications in neurodegenerative processes.

Nonenzymatic protein modifications are generated from direct oxidation of amino acid side chains and from reaction of the nucleophilic side chains of specific amino acids with reactive carbonyl species. These reactions give rise to specific markers that have been analyzed in different neurodegenerative diseases sharing protein aggregation, such as Alzheimer's disease, Pick's disease, Parkinson's disease, dementia with Lewy bodies, Creutzfeldt-Jakob disease, and amyotrophic lateral sclerosis. Collectively, available data demonstrate that oxidative stress homeostasis, mitochondrial function, and energy metabolism are key factors in determining the disease-specific pattern of protein molecular damage. In addition, these findings suggest the lack of a "gold marker of oxidative stress," and, consequently, they strengthen the need for a molecular dissection of the nonenzymatic reactions underlying neurodegenerative processes.

Oxidative and endoplasmic reticulum stress interplay in sporadic amyotrophic lateral sclerosis.

The occurrence of endoplasmic reticulum (ER) stress in the sporadic form of amyotrophic lateral sclerosis (ALS) is unknown, despite it has been recently documented in experimental models of the familial form. Here we show that spinal cord from patients with sporadic ALS showed signs of ER stress, such as increased levels of ER chaperones such as protein-disulfide isomerase, and increased phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha). Among the potential causes of such ER stress proteasomal impairment was confirmed in the same samples by demonstrating increased ubiquitin immunoreactivity and increased protein lipoxidative (125%), glycoxidative (55%) and direct oxidative damage (62%) over control values, as evidenced by mass-spectrometry and immunological methods. We found that protein oxidative damage was strongly associated to ALS-specific changes in fatty acid concentrations, specifically of n-3 series (as docosahexaenoic acid), and in the amount of mitochondrial components as respiratory complexes I and III, suggesting a mitochondrial dysfunction leading to increased free radical production. Oxidative stress was also evidenced in frontal cortex, suggesting that this region is affected early in ALS. As those events were partially reproduced by threohydroxyaspartate exposure in organotypic spinal cord cultures, we concluded that changes in fatty acid composition, mitochondrial function and proteasome activity, which may be driven by excitotoxicity, lead to oxidative stress and finally contribute to ER stress in sporadic ALS.

Mitochondrial dysfunction and oxidative and endoplasmic reticulum stress in argyrophilic grain disease.

Argyrophilic grain disease (AGD) is characterized by the accumulation of hyperphosphorylated 4R tau in dendritic varicosities (i.e."grains") in neurons and pretangles in certain areas of the cerebral cortex and other brain regions. We investigated oxidative and endoplasmic reticulum (ER) stress and dysregulation of mitochondrialbiogenesis as potential mechanisms involved in the AGD pathogenesis. Samples from AGD patients (n = 8) and nonpathologic, age-matched controls (n = 5) were compared using biochemical and immunohistochemical techniques with a panel of antibodies to markers of ER stress responses, stress chaperones, oxidative stress and associated cellular responses, respiratory chain complexes, mitochondrial regulators, and modulators of mitochondrial biogenesis. Because AGD is often associated with other tauopathies, mainly Alzheimer disease (AD), results were also compared with those of a group of similar Braak AD stage cases without grains (n = 5). In both AD and AGD cases, we found activation of key molecules that are involved in the unfolded protein response and lead to elevated ER chaperone levels, increased oxidative stress damage, mainly related to lipoxidation and targeting glycolytic enzymes. Altered expression of components of the respiratory chain markers modulating mitochondrial biogenesis were selectively affected in AGD. The findings suggest that, despite the common pathogenic trends in AD and AGD, there is molecular specificity for AGD.

Depletion of oxidative and endoplasmic reticulum stress regulators in Pick disease.

Both oxidative and endoplasmic reticulum (ER) stress is associated with multiple neurodegenerative, age-related diseases. The rare disorder Pick disease (PiD) shares some pathological hallmarks of other neurodegenerative diseases that may be related to oxidative stress. Importantly, activation of an ER stress response, which is also involved in aging, has not yet been investigated in PiD. In this study, we assessed the implication of ER stress associated with oxidative stress in PiD as a potential mechanism involved in its pathogenesis. Samples from morphologically affected frontal cortex and apparently pathologically preserved occipital cortex showed region-dependent increases in different protein oxidative damage pathways. The oxidative modifications targeted antioxidant enzymes, proteases, heat shock proteins, and synaptic proteins. These effects were associated with compromised proteasomal function and ER stress in frontal cortex samples. In addition, we observed a depletion in ER chaperones (glucose-regulated proteins Grp78/BiP and glucose-regulated protein 94) and differences in tissue content and distribution of nuclear factor-erythroid 2 p45-related respiratory 2, required for cell survival during the unfolded protein response. These results demonstrate increased region-specific protein oxidative damage in PiD, with proteasomal alteration and dysfunctional ER stress response. We suggest this was caused by complete and specific depletion of Grp78/BiP, contributing to the pathophysiology of this neurodegenerative disease.