Crystal structures reveal transient PERK luminal domain tetramerization in endoplasmic reticulum stress signaling

Stress caused by accumulation of misfolded proteins within the endoplasmic reticulum (ER) elicits a cellular unfolded protein response (UPR) aimed at maintaining protein‐folding capacity. PERK, a key upstream component, recognizes ER stress via its luminal sensor/transducer domain, but the molecular events that lead to UPR activation remain unclear. Here, we describe the crystal structures of mammalian PERK luminal domains captured in dimeric state as well as in a novel tetrameric state. Small angle X‐ray scattering analysis (SAXS) supports the existence of both crystal structures also in solution. The salient feature of the tetramer interface, a helix swapped between dimers, implies transient association. Moreover, interface mutations that disrupt tetramer formation in vitro reduce phosphorylation of PERK and its target eIF2α in cells. These results suggest that transient conversion from dimeric to tetrameric state may be a key regulatory step in UPR activation.     

The 12‐15‐lipoxygenase is a modulator of Alzheimer's‐related tau pathology in vivo

12/15‐lipoxygenase (12‐15LO) is a lipid‐peroxidizing enzyme widely expressed in the central nervous system where it has been involved in the neurobiology of Alzheimer's disease (AD) because it modulates amyloid beta (Aβ) and APP processing. However, its biological effect on tau protein is unknown. We investigated the effect of 12‐15LO on tau levels and metabolism in vivo and in vitro and the mechanism involved by using genetic and pharmacologic approaches. While no significant differences were observed in the levels of total tau for both groups, compared with controls, Tg2576 mice overexpressing 12‐15LO had elevated levels of phosphorylated tau at two specific epitopes, Ser 202/Thr 205 and Ser 396. In vitro and in vivo studies show that 12‐15LO modulates tau metabolism specifically via the cdk5 kinase pathway. Associated with these changes were biochemical markers of synaptic pathology. Finally, 12‐15LO‐dependent alteration of tau metabolism was independent from an effect on Aβ. Our findings reveal a novel pathway by which 12‐15LO modulates endogenous tau metabolism making this protein an appealing pharmacologic target for treatment of AD and related tauopathies.     

A novel function for proSAAS as an amyloid anti‐aggregant in Alzheimer's disease

Neurodegenerative diseases such as Alzheimer's disease (AD) are characterized by an abnormal aggregation of misfolded beta‐sheet rich proteins such as β‐amyloid (Aβ). Various ubiquitously expressed molecular chaperones control the correct folding of cellular proteins and prevent the accumulation of harmful species. We here describe a novel anti‐aggregant chaperone function for the neuroendocrine protein proSAAS, an abundant secretory polypeptide that is widely expressed within neural and endocrine tissues and which has previously been associated with neurodegenerative disease in various proteomics studies. In the brains of 12‐month‐old APdE9 mice, and in the cortex of a human AD‐affected brain, proSAAS immunoreactivity was highly colocalized with amyloid pathology. Immunoreactive proSAAS co‐immunoprecipitated with Aβ immunoreactivity in lysates from APdE9 mouse brains. In vitro, proSAAS efficiently prevented the fibrillation of Aβ_1–42 at molar ratios of 1 : 10, and this anti‐aggregation effect was dose dependent. Structure‐function studies showed that residues 97–180 were sufficient for the anti‐aggregation function against Aβ. Finally, inclusion of recombinant proSAAS in the medium of Neuro2a cells, as well as lentiviral‐mediated proSAAS over‐expression, blocked the neurocytotoxic effect of Aβ_1–42 in Neuro2a cells. Taken together, our results suggest that proSAAS may play a role in Alzheimer's disease pathology.

     

Structural characterization of α‐synuclein in an aggregation prone state

The relation of α‐synuclein (αS) aggregation to Parkinson's disease has long been recognized, but the pathogenic species and its molecular properties have yet to be identified. To obtain insight into the properties of αS in an aggregation‐prone state, we studied the structural properties of αS at acidic pH using NMR spectroscopy and computation. NMR demonstrated that αS remains natively unfolded at lower pH, but secondary structure propensities were changed in proximity to acidic residues. The ensemble of conformations of αS at acidic pH is characterized by a rigidification and compaction of the Asp and Glu‐rich C‐terminal region, an increased probability for proximity between the NAC‐region and the C‐terminal region and a lower probability for interactions between the N‐ and C‐terminal regions.     

Neurotrophic factor therapy for nervous system degenerative diseases

The ability of neurotrophic factors to regulate developmental neuronal survival and adult nervous system planticity suggests the use of these molecuales to treat neurodegeneration associated with human diseases. Solid rationales exist for the use of NGF and neurotrophin-3 in the treatment of neuropathies of the peripheral sensory system, insulin-like growth factor and ciliary neurotrophic factor in motor neuron atrophy, and NGF in Alzheimer's disease. Growth factors have been identified for neurons affected in Parkinson's disease, Huntington's disease, and acute brain and spinal cord injury. Various strategies are actively pursued to deliver neurotrophic factors to the brain, and develop therapeutically useful molecules that mimic neurotrophic factor actions or stimulate their production or receptor mechanisms. 1994 John Wiley & Sons, Inc.     

Similarities in the thermodynamics and kinetics of aggregation of disease-related Abeta(1-40) peptides

Increasing evidence indicates that polypeptide aggregation often involves a nucleation and a growth phase, although the relationship between the factors that determine these two phases has not yet been fully clarified. We present here an analysis of several mutations at different sites of the Abeta(1-40) peptide, including those associated with early onset forms of the Alzheimer's disease, which reveals that the effects of specific amino acid substitutions in the sequence of this peptide are strongly modulated by their structural context. Nevertheless, mutations at different positions perturb in a correlated manner the free energies of aggregation as well as the lag times and growth rates. We show that these observations can be rationalized in terms of the intrinsic propensities for aggregation of the Abeta(1-40) sequence, thus suggesting that, in the case of this peptide, the determinants of the thermodynamics and of the nucleation and growth of the aggregates have a similar physicochemical basis.     

Mesenchymal stem cells therapy exerts neuroprotection in a progressive animal model of Parkinson's disease

Parkinson's disease is a common progressive neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra. We investigated whether cell therapy with human mesenchymal stem cells (hMSCs) had a protective effect on progressive dopaminergic neuronal loss in vitro and in vivo. In primary mesencephalic cultures, hMSCs treatment significantly decreased MG-132-induced dopaminergic neuronal loss with a significant reduction of caspase-3 activity. In rats received systemic injection of MG-132, hMSCs treatment in MG-132-treated rats dramatically reduced the decline in the number of tyrosine hydroxylase (TH)-immunoreactive cells, showing an approximately 50% increase in the survival of TH-immunoreactive cells in the substantia nigra compared with the MG-132-treated group. Additionally, hMSC treatment significantly decreased OX-6 immunoreactivity and caspase-3 activity. Histological analysis showed that the number of NuMA-positive cells was 1.7% of total injected hMSCs and 35.7% of these cells were double-stained with NuMA and TH. Adhesive-removal test showed that hMSCs administration in MG-132-treated rats had a tendency to decrease in the mean removal time. This study demonstrates that hMSCs treatment had a protective effect on progressive loss of dopaminergic neurons induced by MG-132 in vitro and in vivo. Complex mechanisms mediated by trophic effects of hMSCs and differentiation of hMSCs into functional TH-immunoreactive neurons may work in the neuroprotective process.     

A freeze-and-thaw-induced fragment of the microtubule-associated protein tau in rat brain extracts: implications for the biochemical assessment of neurotoxicity

Tau is a microtubule-associated protein (MAP) responsible for controlling the stabilization of microtubules in neurons. Tau function is regulated by phosphorylation. However, in some neurological diseases Tau becomes aberrantly hyperphosphorylated, which contributes to the pathogenesis of neurological diseases, known as tauopathies. Western blotting (WB) has been widely employed to determine Tau levels in neurological disease models. However, Tau quantification by WB should be interpreted with care, as this approach has been recognized as prone to produce artifactual results if not properly performed. In the present study, our goal was to evaluate the influence of a freeze-and-thaw cycle, a common procedure preceding WB, to the integrity of Tau in brain homogenates from rats, 3xTg-AD mice and human samples. Homogenates were prepared in ice-cold RIPA buffer supplemented with protease/phosphatase inhibitors. Immediately after centrifugation, an aliquot of the extracts was analyzed via WB to quantify total and phosphorylated Tau levels. The remaining aliquots of the same extracts were stored for at least 2 weeks at either −20 or −80°C and then subjected to WB. Extracts from rodent brains submitted to freeze-and-thaw presented a ∼25 kDa fragment immunoreactive to anti-Tau antibodies. An in-gel digestion followed by mass spectrometry (MS) analysis in excised bands revealed this ∼25 kDa species corresponds to a Tau fragment. Freeze-and-thaw-induced Tau proteolysis was detected even when extracts were stored at −80°C. This phenomenon was not observed in human samples at any storage condition tested. Based on these findings, we strongly recommend the use of fresh extracts of brain samples in molecular analysis of Tau levels in rodents.     

Calcium signaling in Alzheimer's disease & therapies

Alzheimer's disease (AD) is the most common type of dementia and is characterized by the accumulation of amyloid (Aβ) plaques and neurofibrillary tangles in the brain. Much attention has been given to develop AD treatments based on the amyloid cascade hypothesis; however, none of these drugs had good efficacy at improving cognitive functions in AD patients suggesting that Aβ might not be the disease origin. Thus, there are urgent needs for the development of new therapies that target on the proximal cause of AD. Cellular calcium (Ca²⁺) signals regulate important facets of neuronal physiology. An increasing body of evidence suggests that age-related dysregulation of neuronal Ca²⁺ homeostasis may play a proximal role in the pathogenesis of AD as disrupted Ca²⁺ could induce synaptic deficits and promote the accumulation of Aβ plaques and neurofibrillary tangles. Given that Ca²⁺ disruption is ubiquitously involved in all AD pathologies, it is likely that using chemical agents or small molecules specific to Ca²⁺ channels or handling proteins on the plasma membrane and membranes of intracellular organelles to correct neuronal Ca²⁺ dysregulation could open up a new approach to AD prevention and treatment. This review summarizes current knowledge on the molecular mechanisms linking Ca²⁺ dysregulation with AD pathologies and discusses the possibility of correcting neuronal Ca²⁺ disruption as a therapeutic approach for AD.     

TGF-β1 is an organizer of responses to neurodgeneration

TGF-β1 mRNA and protein were recently found to increase in animal brains after experimental lesions that cause local deafferentation or neuron death. Elevations of TGF-β1 mRNA after lesions are prominent in microglia but are also observed in neurons and astrocytes. Moreover, TGF-β1 mRNA autoinduces its own mRNA in the brain. These responses provide models for studying the increases of TGF-β1 protein observed in βA/amyloid-containing extracellular plaques of Alzheimer's disease (AD) and Down's syndrome (DS) and in brain cells of AIDS victims. Involvement of TGF-β1 in these human brain disorders is discussed in relation to the potent effects of TGF-β1 on wound healing and inflammatory responses in peripheral tissues. We hypothesize that TGF-β1 and possibly other TGF-β peptides have organizing roles in responses to neurodegeneration and brain injury that are similar to those observed in non-neural tissues. Work from many laboratories has shown that activities of TGF-β peptides on brain cells include chemotaxis, modification of extracellular matrix, and regulation of cytoskeletal gene expression and of neurotrophins. Similar activities of the TGF-β's are well established in other tissues.     

Neuroprotection by urate on 6‐OHDA‐lesioned rat model of Parkinson's disease: linking to Akt/GSK3β signaling pathway

Higher plasma urate level is reported to be associated with a reduced risk and slower progression of Parkinson's disease (PD). In this study, we explored the effects of urate on dopaminergic neurons in nigrostriatal pathway in the 6‐hydroxydopamine (6‐OHDA) unilaterally lesioned rats. Uric acid (UA), when given twice daily at 200 mg/kg intraperitoneally for 10 consecutive days, elevated urate (the anionic form of UA) in plasma and striatum by 55% and 36.8%, respectively, as compared with vehicle group. This regimen of UA was found to ameliorate the behavioral deficits, dopaminergic neuron loss as well as dopamine depletion in the nigrostriatal system. Moreover, UA administration was capable of increasing glutathione level and superoxide dismutase activity while decreasing malondialdehyde accumulation in striatum. In addition, the phosphorylation of both protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK3β) in the lesioned striata of 6‐OHDA‐lesioned rats was dramatically reduced as compared with sham‐operated rats. This reduction was attenuated in the Parkinsonian rats receiving UA treatment. Similarly, in vitro findings showed that UA alleviated the decrease in Akt activation and the increase in GSK3β activity caused by 6‐OHDA. Furthermore, neuroprotection by urate and its regulation on GSK3β phosphorylation at Ser9 was found to be abolished in the presence of PI3K inhibitor. Therefore, our findings demonstrated that urate was able to protect dopaminergic neurons in rat nigrostriatal pathway against the neurotoxicity of 6‐OHDA, and showed that its beneficial effects may be related to its regulation on Akt/GSK3β signaling.     

The putative multidrug resistance protein MRP‐7 inhibits methylmercury‐associated animal toxicity and dopaminergic neurodegeneration in Caenorhabditis elegans

Parkinson's disease (PD) is the most prevalent neurodegenerative motor disorder worldwide, and results in the progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta. Gene–environment interactions are believed to play a significant role in the vast majority of PD cases, yet the toxicants and the associated genes involved in the neuropathology are largely ill‐defined. Recent epidemiological and biochemical evidence suggests that methylmercury (MeHg) may be an environmental toxicant that contributes to the development of PD. Here, we report that a gene coding for the putative multidrug resistance protein MRP‐7 in Caenorhabditis elegans modulates whole animal and DA neuron sensitivity to MeHg. In this study, we demonstrate that genetic knockdown of MRP‐7 results in a twofold increase in Hg levels and a dramatic increase in stress response proteins associated with the endoplasmic reticulum, golgi apparatus, and mitochondria, as well as an increase in MeHg‐associated animal death. Chronic exposure to low concentrations of MeHg induces MRP‐7 gene expression, while exposures in MRP‐7 genetic knockdown animals results in a loss of DA neuron integrity without affecting whole animal viability. Furthermore, transgenic animals expressing a fluorescent reporter behind the endogenous MRP‐7 promoter indicate that the transporter is expressed in DA neurons. These studies show for the first time that a multidrug resistance protein is expressed in DA neurons, and its expression inhibits MeHg‐associated DA neuron pathology.