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1.  A comprehensive multiomics approach toward understanding the relationship between aging and dementia 
Aging (Albany NY)  2015;7(11):937-952.
Because age is the greatest risk factor for sporadic Alzheimer's disease (AD), phenotypic screens based upon old age-associated brain toxicities were used to develop the potent neurotrophic drug J147. Since certain aspects of aging may be primary cause of AD, we hypothesized that J147 would be effective against AD-associated pathology in rapidly aging SAMP8 mice and could be used to identify some of the molecular contributions of aging to AD. An inclusive and integrative multiomics approach was used to investigate protein and gene expression, metabolite levels, and cognition in old and young SAMP8 mice. J147 reduced cognitive deficits in old SAMP8 mice, while restoring multiple molecular markers associated with human AD, vascular pathology, impaired synaptic function, and inflammation to those approaching the young phenotype. The extensive assays used in this study identified a subset of molecular changes associated with aging that may be necessary for the development of AD.
PMCID: PMC4694064  PMID: 26564964
aging; Alzheimer's disease; multiomics; SAMP8 mice; inflammation; J147
2.  Back to the Future with Phenotypic Screening 
ACS Chemical Neuroscience  2014;5(7):503-513.
There are no disease-modifying drugs for any old age associated neurodegenerative disease or stroke. This is at least in part due to the failure of drug developers to recognize that the vast majority of neurodegenerative diseases arise from a confluence of multiple toxic insults that accumulate during normal aging and interact with genetic and environmental risk factors. Thus, it is unlikely that the current single target approach based upon rare dominant mutations or even a few preselected targets is going to yield useful drugs for these conditions. Therefore, the identification of drug candidates for neurodegeneration should be based upon their efficacy in phenotypic screening assays that reflect the biology of the aging brain, not a single, preselected target. It is argued here that this approach to drug discovery is the most likely to produce safe and effective drugs for neurodegenerative diseases.
doi:10.1021/cn500051h
PMCID: PMC4102969  PMID: 24902068
Phenotypic screening; Alzheimer’s; neurodegeneration; drug discovery; medicinal chemistry; cell culture; aging
3.  Modulation of p25 and Inflammatory Pathways by Fisetin Maintains Cognitive Function in Alzheimer’s Disease Transgenic Mice 
Aging cell  2013;13(2):379-390.
Alzheimer’s disease (AD) is the most common type of dementia. It is the only one of the top ten causes of death in the US for which prevention strategies have not been developed. Although AD has traditionally been associated with the deposition of amyloid β plaques and tau tangles, it is becoming increasingly clear that it involves disruptions in multiple cellular systems. Therefore, it is unlikely that hitting a single target will result in significant benefits to patients with AD. An alternative approach is to identify molecules that have multiple biological activities that are relevant to the disease. Fisetin is a small, orally active molecule which can act on many of the target pathways implicated in AD. We show here that oral administration of fisetin to APPswe/PS1dE9 double transgenic AD mice from 3 to 12 months of age prevents the development of learning and memory deficits. This correlates with an increase in ERK phosphorylation along with a decrease in protein carbonylation, a marker of oxidative stress. Importantly, fisetin also reduces the levels of the cyclin dependent kinase 5 (Cdk5) activator p35 cleavage product, p25, in both control and AD brains. Elevated levels of p25 relative to p35 cause dysregulation of Cdk5 activity leading to neuroinflammation and neurodegeneration. These fisetin-dependent changes correlate with additional anti-inflammatory effects, including alterations in global eicosanoid synthesis, and the maintenance of markers of synaptic function in the AD mice. Together, these results suggest that fisetin may provide a new approach to the treatment of AD.
doi:10.1111/acel.12185
PMCID: PMC3954948  PMID: 24341874
oxidative stress; ERK; eicosanoid; astrogliosis; prostaglandin; LOX
4.  Crossreactivity of a human autoimmune TCR is dominated by a single TCR loop 
Nature communications  2013;4:2623.
Self-reactive CD4 T cells are thought to have a central role in the pathogenesis of many chronic inflammatory human diseases. Microbial peptides can activate self-reactive T cells, but the structural basis for such crossreactivity is not well understood. The Hy.1B11 T cell receptor (TCR) originates from a patient with multiple sclerosis and recognizes the self-antigen myelin basic protein. Here we report the structural mechanism of TCR crossreactivity with two distinct peptides from human pathogens. The structures show that a single TCR residue (CDR3α F95) makes the majority of contacts with the self-peptide and both microbial peptides (66.7–80.6%) due to a highly tilted TCR-binding topology on the peptide-MHC surface. Further, a neighbouring residue located on the same TCR loop (CDR3α E98) forms an energetically critical interaction with the MHC molecule. These data show how binding by a self-reactive TCR favors crossreactivity between self and microbial antigens.
doi:10.1038/ncomms3623
PMCID: PMC4193804  PMID: 24136005
5.  Modulation of p25 and inflammatory pathways by fisetin maintains cognitive function in Alzheimer’s disease transgenic mice 
Aging Cell  2013;13(2):379-390.
Alzheimer’s disease (AD) is the most common type of dementia. It is the only one of the top ten causes of death in the USA for which prevention strategies have not been developed. Although AD has traditionally been associated with the deposition of amyloid β plaques and tau tangles, it is becoming increasingly clear that it involves disruptions in multiple cellular systems. Therefore, it is unlikely that hitting a single target will result in significant benefits to patients with AD. An alternative approach is to identify molecules that have multiple biological activities that are relevant to the disease. Fisetin is a small, orally active molecule which can act on many of the target pathways implicated in AD. We show here that oral administration of fisetin to APPswe/PS1dE9 double transgenic AD mice from 3 to 12 months of age prevents the development of learning and memory deficits. This correlates with an increase in ERK phosphorylation along with a decrease in protein carbonylation, a marker of oxidative stress. Importantly, fisetin also reduces the levels of the cyclin-dependent kinase 5 (Cdk5) activator p35 cleavage product, p25, in both control and AD brains. Elevated levels of p25 relative to p35 cause dysregulation of Cdk5 activity leading to neuroinflammation and neurodegeneration. These fisetin-dependent changes correlate with additional anti-inflammatory effects, including alterations in global eicosanoid synthesis, and the maintenance of markers of synaptic function in the AD mice. Together, these results suggest that fisetin may provide a new approach to the treatment of AD.
doi:10.1111/acel.12185
PMCID: PMC3954948  PMID: 24341874
astrogliosis; ERK; eicosanoid; lipoxygenase; oxidative stress; prostaglandin
6.  Modulation of 5-Lipoxygenase in Proteotoxicity and Alzheimer's Disease 
The Journal of Neuroscience  2013;33(25):10512-10525.
The accumulation of intracellular β amyloid (Aβ) may be one of the factors leading to neuronal cell death in Alzheimer's disease (AD). Using a pyrazole called CNB-001, which was selected for its ability to reduce intracellular Aβ, we show that the activation of the eIF2α/ATF4 arm of the unfolded protein response is sufficient to degrade aggregated intracellular Aβ. CNB-001 is a potent inhibitor of 5-lipoxygenase (5-LOX), decreases 5-LOX expression, and increases proteasome activity. 5-LOX inhibition induces eIF2α and PERK (protein kinase R-like extracellular signal-regulated kinase) phosphorylation, and HSP90 and ATF4 levels. When fed to AD transgenic mice, CNB-001 also increases eIF2α phosphorylation and HSP90 and ATF4 levels, and limits the accumulation of soluble Aβ and ubiquitinated aggregated proteins. Finally, CNB-001 maintains the expression of synapse-associated proteins and improves memory. Therefore, 5-LOX metabolism is a key element in the promotion of endoplasmic reticulum dysfunction, and its inhibition under conditions of stress is sufficient to reduce proteotoxicity both in vivo and in vitro.
doi:10.1523/JNEUROSCI.5183-12.2013
PMCID: PMC3685841  PMID: 23785163
7.  Diabetes exacerbates amyloid and neurovascular pathology in aging-accelerated mice 
Aging cell  2012;11(6):1017-1026.
Summary
Mounting evidence supports a link between diabetes, cognitive dysfunction and aging. However, the physiological mechanisms by which diabetes impacts brain function and cognition are not fully understood. To determine how diabetes contributes to cognitive dysfunction and age-associated pathology, we used streptozotocin to induce type 1 diabetes (T1D) in senescence-accelerated prone 8 (SAMP8) and senescence-resistant 1 (SAMR1) mice. Contextual fear conditioning demonstrated that T1D resulted in the development of cognitive deficits in SAMR1 mice similar to those seen in age-matched, non-diabetic SAMP8 mice. No further cognitive deficits were observed when the SAMP8 mice were made diabetic. T1D dramatically increased Aβ and glial fibrillary acidic protein (GFAP) immunoreactivity in the hippocampus of SAMP8 mice and to a lesser extent in age-matched SAMR1 mice. Further analysis revealed aggregated Aβ within astrocyte processes surrounding vessels. Western blot analyses from T1D SAMP8 mice showed elevated APP processing and protein glycation along with increased inflammation. T1D elevated tau phosphorylation in the SAMR1 mice but did not further increase it in the SAMP8 mice where it was already significantly higher. These data suggest that aberrant glucose metabolism potentiates the aging phenotype in old mice and contributes to early stage CNS pathology in younger animals.
doi:10.1111/acel.12002
PMCID: PMC3500443  PMID: 22938075
Aging; Diabetes; Alzheimer’s disease; Neurovascular inflammation; Amyloid angiopathy; Advanced glycation end-products (AGEs)
8.  Local changes in lipid environment of TCR microclusters regulate membrane binding by the CD3ε cytoplasmic domain 
The Journal of Experimental Medicine  2012;209(13):2423-2439.
The CD3ε and ζ cytoplasmic domains of the T cell receptor bind to the inner leaflet of the plasma membrane (PM), and a previous nuclear magnetic resonance structure showed that both tyrosines of the CD3ε immunoreceptor tyrosine-based activation motif partition into the bilayer. Electrostatic interactions between acidic phospholipids and clusters of basic CD3ε residues were previously shown to be essential for CD3ε and ζ membrane binding. Phosphatidylserine (PS) is the most abundant negatively charged lipid on the inner leaflet of the PM and makes a major contribution to membrane binding by the CD3ε cytoplasmic domain. Here, we show that TCR triggering by peptide–MHC complexes induces dissociation of the CD3ε cytoplasmic domain from the plasma membrane. Release of the CD3ε cytoplasmic domain from the membrane is accompanied by a substantial focal reduction in negative charge and available PS in TCR microclusters. These changes in the lipid composition of TCR microclusters even occur when TCR signaling is blocked with a Src kinase inhibitor. Local changes in the lipid composition of TCR microclusters thus render the CD3ε cytoplasmic domain accessible during early stages of T cell activation.
doi:10.1084/jem.20120790
PMCID: PMC3526357  PMID: 23166358
9.  The neurotrophic compound J147 reverses cognitive impairment in aged Alzheimer's disease mice 
Introduction
Despite years of research, there are no disease-modifying drugs for Alzheimer's disease (AD), a fatal, age-related neurodegenerative disorder. Screening for potential therapeutics in rodent models of AD has generally relied on testing compounds before pathology is present, thereby modeling disease prevention rather than disease modification. Furthermore, this approach to screening does not reflect the clinical presentation of AD patients which could explain the failure to translate compounds identified as beneficial in animal models to disease modifying compounds in clinical trials. Clearly a better approach to pre-clinical drug screening for AD is required.
Methods
To more accurately reflect the clinical setting, we used an alternative screening strategy involving the treatment of AD mice at a stage in the disease when pathology is already advanced. Aged (20-month-old) transgenic AD mice (APP/swePS1ΔE9) were fed an exceptionally potent, orally active, memory enhancing and neurotrophic molecule called J147. Cognitive behavioral assays, histology, ELISA and Western blotting were used to assay the effect of J147 on memory, amyloid metabolism and neuroprotective pathways. J147 was also investigated in a scopolamine-induced model of memory impairment in C57Bl/6J mice and compared to donepezil. Details on the pharmacology and safety of J147 are also included.
Results
Data presented here demonstrate that J147 has the ability to rescue cognitive deficits when administered at a late stage in the disease. The ability of J147 to improve memory in aged AD mice is correlated with its induction of the neurotrophic factors NGF (nerve growth factor) and BDNF (brain derived neurotrophic factor) as well as several BDNF-responsive proteins which are important for learning and memory. The comparison between J147 and donepezil in the scopolamine model showed that while both compounds were comparable at rescuing short term memory, J147 was superior at rescuing spatial memory and a combination of the two worked best for contextual and cued memory.
Conclusion
J147 is an exciting new compound that is extremely potent, safe in animal studies and orally active. J147 is a potential AD therapeutic due to its ability to provide immediate cognition benefits, and it also has the potential to halt and perhaps reverse disease progression in symptomatic animals as demonstrated in these studies.
doi:10.1186/alzrt179
PMCID: PMC3706879  PMID: 23673233
10.  Chemical Modification of the Multi-Target Neuroprotective Compound Fisetin 
Journal of Medicinal Chemistry  2011;55(1):378-389.
Many factors are implicated in age-related CNS disorders making it unlikely that modulating only a single factor will provide effective treatment. Perhaps a better approach is to identify small molecules that have multiple biological activities relevant to the maintenance of brain function. Recently, we identified an orally active, neuroprotective and cognition-enhancing molecule, the flavonoid fisetin, that is effective in several animal models of CNS disorders. Fisetin has direct antioxidant activity and can also increase the intracellular levels of glutathione (GSH), the major endogenous antioxidant. In addition, fisetin has both neurotrophic and anti-inflammatory activity. However, its relatively high EC50 in cell based assays, low lipophilicity, high tPSA and poor bioavailability suggest that there is room for medicinal chemical improvement. Here we describe a multi-tiered approach to screening that has allowed us to identify fisetin derivatives with significantly enhanced activity in an in vitro neuroprotection model while at the same time maintaining other key activities.
doi:10.1021/jm2012563
PMCID: PMC3291199  PMID: 22192055
11.  Self-reactive human CD4 T cell clones form unusual immunological synapses 
Compared with influenza-specific T cells, self-reactive T cells from patients with multiple sclerosis or type 1 diabetes fail to slow down and do not form normal immunological synapses upon encounter with cognate self-peptide presented by MHC.
Recognition of self–peptide-MHC (pMHC) complexes by CD4 T cells plays an important role in the pathogenesis of many autoimmune diseases. We analyzed formation of immunological synapses (IS) in self-reactive T cell clones from patients with multiple sclerosis and type 1 diabetes. All self-reactive T cells contained a large number of phosphorylated T cell receptor (TCR) microclusters, indicative of active TCR signaling. However, they showed little or no visible pMHC accumulation or transport of TCR–pMHC complexes into a central supramolecular activation cluster (cSMAC). In contrast, influenza-specific T cells accumulated large quantities of pMHC complexes in microclusters and a cSMAC, even when presented with 100-fold lower pMHC densities. The self-reactive T cells also maintained a high degree of motility, again in sharp contrast to virus-specific T cells. 2D affinity measurements of three of these self-reactive T cell clones demonstrated a normal off-rate but a slow on-rate of TCR binding to pMHC. These unusual IS features may facilitate escape from negative selection by self-reactive T cells encountering very small amounts of self-antigen in the thymus. However, these same features may enable acquisition of effector functions by self-reactive T cells encountering large amounts of self-antigen in the target organ of the autoimmune disease.
doi:10.1084/jem.20111485
PMCID: PMC3280872  PMID: 22312112
12.  p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects 
Autophagy  2011;7(6):572-583.
Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.
doi:10.4161/auto.7.6.14943
PMCID: PMC3127048  PMID: 21325881
p62; Ref(2)P; insoluble ubiquitinated proteins; aggregates; neural degeneration; Alzheimer disease; aging; macroautophagy
13.  De-Risking of Stilbazulenyl Nitrone (STAZN), a Lipophilic Nitrone to Treat Stroke Using a Unique Panel of In Vitro Assays 
Translational stroke research  2011;2(2):209-217.
In the present study, we used a comprehensive panel of in vitro assays to evaluate the efficacy and safety of stilbazulenyl nitrone (STAZN) as a lead compound to treat acute ischemic stroke. First, we measured neuroprotection in vitro using two different HT22 hippocampal nerve cell assays. Secondly, to de-risk drug development, we used CeeTox analysis with the H4IIE rat hepatoma cell line to determine the acute toxicity profile of STAZN. Third, STAZN was tested in microsomes from four species for measures of metabolic stability. Last, we determined the Ames test genotoxicity profile of STAZN using Salmonella typhimurium TA989 and TA100. In vitro, STAZN was neuroprotective against toxicity induced by iodoacetic acid, and oxytosis-induced glutathione depletion was initiated by glutamate, with an EC50 value of 1–5 μM. Secondly, using CeeTox analysis, the estimated CTox value (i.e., sustained concentration expected to produce toxicity in a rat 14-day repeat dose study) for STAZN was calculated to be 260 μM. Third, the half-life of STAZN in humans, dogs, and rats was 60–78 min. Last, the genotoxicity profile showed that STAZN did not induce bacterial colony growth under any conditions tested, indicating the lack of mutagenicity with this compound. STAZN appears to be a multi-target neuroprotective compound that has an excellent safety profile in both the CeeTox and Ames mutagenicity assays. STAZN may have significant potential as a novel neuroprotective agent to treat stroke and should be pursued in clinically relevant embolic stroke models.
doi:10.1007/s12975-011-0071-7
PMCID: PMC3191534  PMID: 22003372
Antioxidant; Nitrone; Safety; Efficacy; Genotoxicity; CeeTox
14.  Brain and Spinal Cord Interaction: A Dietary Curcumin Derivative Counteracts Locomotor and Cognitive Deficits After Brain Trauma 
Background
In addition to cognitive dysfunction, locomotor deficits are prevalent in traumatic brain injured (TBI) patients; however, it is unclear how a concussive injury can affect spinal cord centers. Moreover, there are no current efficient treatments that can counteract the broad pathology associated with TBI.
Objective
The authors have investigated potential molecular basis for the disruptive effects of TBI on spinal cord and hippocampus and the neuroprotection of a curcumin derivative to reduce the effects of experimental TBI.
Methods
The authors performed fluid percussion injury (FPI) and then rats were exposed to dietary supplementation of the curcumin derivative (CNB-001; 500 ppm). The curry spice curcumin has protective capacity in animal models of neurodegenerative diseases, and the curcumin derivative has enhanced brain absorption and biological activity.
Results
The results show that FPI in rats, in addition to reducing learning ability, reduced locomotor performance. Behavioral deficits were accompanied by reductions in molecular systems important for synaptic plasticity underlying behavioral plasticity in the brain and spinal cord. The post-TBI dietary supplementation of the curcumin derivative normalized levels of BDNF, and its downstream effectors on synaptic plasticity (CREB, synapsin I) and neuronal signaling (CaMKII), as well as levels of oxidative stress–related molecules (SOD, Sir2).
Conclusions
These studies define a mechanism by which TBI can compromise centers related to cognitive processing and locomotion. The findings also show the influence of the curcumin derivative on synaptic plasticity events in the brain and spinal cord and emphasize the therapeutic potential of this noninvasive dietary intervention for TBI.
doi:10.1177/1545968310397706
PMCID: PMC3258099  PMID: 21343524
traumatic brain injury; hippocampus; learning; BDNF; curcumin derivative
15.  Delayed Treatment with a Novel Neurotrophic Compound Reduces Behavioral Deficits in Rabbit Ischemic Stroke 
Journal of neurochemistry  2010;116(1):122-131.
Acute ischemic stroke is a major risk for morbidity and mortality in our aging population. Currently only one drug, the thrombolytic tissue plasminogen activator, is approved by the FDA to treat stroke. Therefore, there is a need to develop novel drugs that promote neuronal survival following stroke. We have synthesized a novel neuroprotective molecule called CNB-001 that has neurotrophic activity, enhances memory, and blocks cell death in multiple toxicity assays related to ischemic stroke. In this study, we tested the efficacy of CNB-001 in a rigorous rabbit ischemic stroke model and determined the molecular basis of its in vivo activity. CNB-001 has substantial beneficial properties in an in vitro ischemia assay and improves the behavioral outcome of rabbit ischemic stroke even when administered 1 h after the insult, a therapeutic window in this model comparable to tissue plasminogen activator. In addition, we elucidated the protein kinase pathways involved in neuroprotection. CNB-001 maintains the calcium-calmodulin-dependent kinase signaling pathways associated with neurotrophic growth factors that are critical for the maintenance of neuronal function. On the basis of its in vivo efficacy and novel mode of action, we conclude that CNB-001 has a great potential for the treatment of ischemic stroke as well as other CNS pathologies.
doi:10.1111/j.1471-4159.2010.07090.x
PMCID: PMC3004475  PMID: 21054387
ischemia; trophic factor; neuroprotection; models; drugs; protein kinases
16.  A Novel Neurotrophic Drug for Cognitive Enhancement and Alzheimer's Disease 
PLoS ONE  2011;6(12):e27865.
Currently, the major drug discovery paradigm for neurodegenerative diseases is based upon high affinity ligands for single disease-specific targets. For Alzheimer's disease (AD), the focus is the amyloid beta peptide (Aß) that mediates familial Alzheimer's disease pathology. However, given that age is the greatest risk factor for AD, we explored an alternative drug discovery scheme that is based upon efficacy in multiple cell culture models of age-associated pathologies rather than exclusively amyloid metabolism. Using this approach, we identified an exceptionally potent, orally active, neurotrophic molecule that facilitates memory in normal rodents, and prevents the loss of synaptic proteins and cognitive decline in a transgenic AD mouse model.
doi:10.1371/journal.pone.0027865
PMCID: PMC3237323  PMID: 22194796
17.  Errors in text 
doi:10.1258/jrsm.2008.08k016
PMCID: PMC2587391  PMID: 18779242
18.  A highly tilted binding mode by a self-reactive T cell receptor results in altered engagement of peptide and MHC 
A TCR derived from a patient with relapsing-remitting multiple sclerosis engages the self-peptide myelin basic protein in the context of HLA-DQ1 in a very unusual way.
Self-reactive T cells that escape elimination in the thymus can cause autoimmune pathology, and it is therefore important to understand the structural mechanisms of self-antigen recognition. We report the crystal structure of a T cell receptor (TCR) from a patient with relapsing-remitting multiple sclerosis that engages its self-peptide–major histocompatibility complex (pMHC) ligand in an unusual manner. The TCR is bound in a highly tilted orientation that prevents interaction of the TCR-α chain with the MHC class II β chain helix. In this structure, only a single germline-encoded TCR loop engages the MHC protein, whereas in most other TCR-pMHC structures all four germline-encoded TCR loops bind to the MHC helices. The tilted binding mode also prevents peptide contacts by the short complementarity-determining region (CDR) 3β loop, and interactions that contribute to peptide side chain specificity are focused on the CDR3α loop. This structure is the first example in which only a single germline-encoded TCR loop contacts the MHC helices. Furthermore, the reduced interaction surface with the peptide may facilitate TCR cross-reactivity. The structural alterations in the trimolecular complex are distinct from previously characterized self-reactive TCRs, indicating that there are multiple unusual ways for self-reactive TCRs to bind their pMHC ligand.
doi:10.1084/jem.20100725
PMCID: PMC3023130  PMID: 21199956
19.  HLA-DM Captures Partially Empty HLA-DR Molecules for Catalyzed Peptide Removal 
Nature immunology  2010;12(1):54-61.
The mechanisms of HLA-DM catalyzed peptide exchange remain uncertain. We found that all stages of the interaction of DM with HLA-DR were dependent on the occupancy state of the peptide binding groove. High-affinity peptides were protected from removal by DM through two mechanisms: peptide binding induced dissociation of a long-lived complex of empty DR and DM, and high-affinity DR-peptide complexes bound DM only very slowly. Non-binding covalent DR-peptide complexes were converted to efficient DM binders upon truncation of an N-terminal peptide segment that emptied the P1 pocket and disrupted conserved hydrogen bonds to MHC. DM thus only binds to DR conformers in which a critical part of the binding site is vacant, due to spontaneous peptide motion.
doi:10.1038/ni.1967
PMCID: PMC3018327  PMID: 21131964
20.  Fisetin Lowers Methylglyoxal Dependent Protein Glycation and Limits the Complications of Diabetes 
PLoS ONE  2011;6(6):e21226.
The elevated glycation of macromolecules by the reactive dicarbonyl and α-oxoaldehyde methylglyoxal (MG) has been associated with diabetes and its complications. We have identified a rare flavone, fisetin, which increases the level and activity of glyoxalase 1, the enzyme required for the removal of MG, as well as the synthesis of its essential co-factor, glutathione. It is shown that fisetin reduces two major complications of diabetes in Akita mice, a model of type 1 diabetes. Although fisetin had no effect on the elevation of blood sugar, it reduced kidney hypertrophy and albuminuria and maintained normal levels of locomotion in the open field test. This correlated with a reduction in proteins glycated by MG in the blood, kidney and brain of fisetin-treated animals along with an increase in glyoxalase 1 enzyme activity and an elevation in the expression of the rate-limiting enzyme for the synthesis of glutathione, a co-factor for glyoxalase 1. The expression of the receptor for advanced glycation end products (RAGE), serum amyloid A and serum C-reactive protein, markers of protein oxidation, glycation and inflammation, were also increased in diabetic Akita mice and reduced by fisetin. It is concluded that fisetin lowers the elevation of MG-protein glycation that is associated with diabetes and ameliorates multiple complications of the disease. Therefore, fisetin or a synthetic derivative may have potential therapeutic use for the treatment of diabetic complications.
doi:10.1371/journal.pone.0021226
PMCID: PMC3124487  PMID: 21738623
21.  Amyloid Beta Resistance in Nerve Cell Lines Is Mediated by the Warburg Effect 
PLoS ONE  2011;6(4):e19191.
Amyloid beta (Aβ) peptide accumulation in the brains of patients with Alzheimer's disease (AD) is closely associated with increased nerve cell death. However, many cells survive and it is important to understand the mechanisms involved in this survival response. Recent studies have shown that an anti-apoptotic mechanism in cancer cells is mediated by aerobic glycolysis, also known as the Warburg effect. One of the major regulators of aerobic glycolysis is pyruvate dehydrogenase kinase (PDK), an enzyme which represses mitochondrial respiration and forces the cell to rely heavily on glycolysis, even in the presence of oxygen. Recent neuroimaging studies have shown that the spatial distribution of aerobic glycolysis in the brains of AD patients strongly correlates with Aβ deposition. Interestingly, clonal nerve cell lines selected for resistance to Aβ exhibit increased glycolysis as a result of activation of the transcription factor hypoxia inducible factor 1. Here we show that Aβ resistant nerve cell lines upregulate Warburg effect enzymes in a manner reminiscent of cancer cells. In particular, Aβ resistant nerve cell lines showed elevated PDK1 expression in addition to an increase in lactate dehydrogenase A (LDHA) activity and lactate production when compared to control cells. In addition, mitochondrial derived reactive oxygen species (ROS) were markedly diminished in resistant but not sensitive cells. Chemically or genetically inhibiting LDHA or PDK1 re-sensitized resistant cells to Aβ toxicity. These findings suggest that the Warburg effect may contribute to apoptotic-resistance mechanisms in the surviving neurons of the AD brain. Loss of the adaptive advantage afforded by aerobic glycolysis may exacerbate the pathophysiological processes associated with AD.
doi:10.1371/journal.pone.0019191
PMCID: PMC3082554  PMID: 21541279
22.  A multi-dimensional Structure-Activity Relationship of a protein in its aggregated states 
Protein aggregates are both associated with disease and function. Because a variety of factors induce protein aggregation, a given protein can aggregate into different states. Here, we compare the structures and activities of five distinct protein aggregates of a single protein. Despite the diverse chemical, physical and biological treatments used to induce aggregation, all aggregate types contain the cross-β-sheet motif. However, they are structurally distinct, having different segments of the protein sequence involved in secondary structure formation. Because of these structural differences each aggregate has a unique set of properties. These include affinity to ATP, Thioflavin T, DNA, and membrane mimics, and interference with cell viability. The key to their multiple properties may be that the repetitive nature of the cross-β-sheet motif guarantees for many potent activities through cooperativity. The observed multidimensional structure-activity relationship of protein aggregates may be important for amyloid diseases but may also be advantageous in nanotechnology.
doi:10.1002/anie.201000068
PMCID: PMC3004770  PMID: 20397175
Protein Aggregation; Amyloid Fibrils; Inclusion Bodies; Cross-β-Sheet; EM; X-ray Diffraction; FT-IR; NMR; Hydrogen/Deuterium Exchange; Structure-Activity
23.  Functional Amyloids as Natural Storage of Peptide Hormones in Pituitary Secretory Granules 
Science (New York, N.Y.)  2009;325(5938):328-332.
Amyloids are highly organized cross β-sheet-rich protein or peptide aggregates that are associated with pathological conditions including Alzheimer’s disease and type II diabetes. However, amyloids may also have a normal biological function as demonstrated by fungal prions, which are involved in prion replication, and the amyloid protein Pmel17, which is involved in mammalian skin pigmentation. Here, we show that peptide and protein hormones in secretory granules of the endocrine system are stored in an amyloid-like cross β-sheet-rich conformation. Thus, in contrast to the original association of amyloids with diseases, functional amyloids in the pituitary and other organs can contribute to normal cell and tissue physiology.
doi:10.1126/science.1173155
PMCID: PMC2865899  PMID: 19541956
24.  Synaptojanin-1 plays a key role in astrogliogenesis: possible relevance for Down’s syndrome 
Cell death and differentiation  2009;16(6):910-920.
There is increasing interest in gliogenesis as the relevance of glia to both brain development and pathology becomes better understood. However, little is known about this process. The use of multidimensional protein identification technology (MudPIT) to identify changes in phosphoprotein levels in rat neural precursor cells treated with cytokines or retinoic acid showed that phosphorylation of the catalytic subunit of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K p110α) and dephosphorylation of the inositol phosphatase synaptojanin-1 were common to the gliogenic stimuli. While PI3K was found to be involved in both neuro- and astrogliogenesis, synaptojanin-1 was specifically involved in astrogliogenesis of neural precursor cells. The role of synaptojanin-1 in astrogliogenesis was further confirmed by analysis of neuron- and glia-specific markers in synaptojanin-1 knockout mouse brain. Additional experiments showed that the Sac1-like phosphatase domain of synaptojanin-1 is the responsible for the observed astrogliogenic effect. Our results strongly indicate that phosphatidylinositol metabolism plays a key role in astrogliogenesis. The relevance of our findings for Down’s syndrome pathology is discussed.
doi:10.1038/cdd.2009.24
PMCID: PMC2807404  PMID: 19282871
synaptojanin-1; gliogenesis; retinoic acid; cytokine; Down’s syndrome
25.  NEURAL CELLS SECRETE A UNIQUE REPERTOIRE OF PROTEINS 
Journal of neurochemistry  2009;109(2):427-435.
Proteins that are released from cells consist of those in the extracellular matrix, as well as extracellular signaling and adhesion molecules. The majority of these extracellular proteins are, however, unknown. To determine their identity, we have used a proteomics approach to define proteins released from neurons, astrocytes and neural precursor cells. Using 2-dimensional gels and liquid chromatography/mass spectrometry technology, it is shown that while astrocytes release a relatively small number of proteins, neurons and neuronal precursor cells release a larger number of proteins with more functional diversity. Although there is overlap between the different cell types, the exact composition of the extracellular protein pool is unique for each cell population. The various subsets of extracellular neural proteins include those involved in cellular Redox regulation and chaperones. In addition, many proteolytic enzymes are found outside of the cell. These data show that the extracellular space within the nervous system has a more diverse protein composition than previously thought.
doi:10.1111/j.1471-4159.2009.05968.x
PMCID: PMC2676922  PMID: 19200335
Neuronal cells; secreted proteins; proteomics; neurons; astrocytes

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