In vivo NMR studies of regional cerebral energetics in MPTP model of Parkinson's disease: recovery of cerebral metabolism with acute levodopa treatment

In this study, we have evaluated cerebral atrophy, neurometabolite homeostasis, and neural energetics in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridin (MPTP) model of Parkinson's disease. In addition, the efficacy of acute l ‐DOPA treatment for the reversal of altered metabolic functions was also evaluated. Cerebral atrophy and neurochemical profile were monitored in vivo using MRI and ¹H MR Spectroscopy. Cerebral energetics was studied by ¹H‐[¹³C]‐NMR spectroscopy in conjunction with infusion of ¹³C labeled [1,6^‐13C₂]glucose or [2‐¹³C]acetate. MPTP treatment led to reduction in paw grip strength and increased level of GABA and myo‐inositol in striatum and olfactory bulb. ¹³C Labeling of glutamate‐C4 (1.93 ± 0.24 vs. 1.48 ± 0.06 μmol/g), GABA‐C2 (0.24 ± 0.04 vs. 0.18 ± 0.02 μmol/g) and glutamaine‐C4 (0.26 ± 0.04 vs. 0.20 ± 0.04 μmol/g) from [1,6‐¹³C₂]glucose was found to be decreased with MPTP exposure in striatum as well as in other brain regions. However, glutamine‐C4 labeling from [2‐¹³C]acetate was found to be increased in the striatum of the MPTP‐treated mice. Acute l ‐DOPA treatment failed to normalize the increased ventricular size and level of metabolites but recovered the paw grip strength and ¹³C labeling of amino acids from [1,6‐¹³C₂]glucose and [2‐¹³C]acetate in MPTP‐treated mice. These data indicate that brain energy metabolism is impaired in Parkinson's disease and acute l ‐DOPA therapy could temporarily recover the cerebral metabolism.

     

Kindlin Binds Migfilin Tandem LIM Domains and Regulates Migfilin Focal Adhesion Localization and Recruitment Dynamics

Focal adhesions (FAs), sites of tight adhesion to the extracellular matrix, are composed of clusters of transmembrane integrin adhesion receptors and intracellular proteins that link integrins to the actin cytoskeleton and signaling pathways. Two integrin-binding proteins present in FAs, kindlin-1 and kindlin-2, are important for integrin activation, FA formation, and signaling. Migfilin, originally identified in a yeast two-hybrid screen for kindlin-2-interacting proteins, is a LIM domain-containing adaptor protein found in FAs and implicated in control of cell adhesion, spreading, and migration. By binding filamin, migfilin provides a link between kindlin and the actin cytoskeleton. Here, using a combination of kindlin knockdown, biochemical pulldown assays, fluorescence microscopy, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP), we have established that the C-terminal LIM domains of migfilin dictate its FA localization, shown that these domains mediate an interaction with kindlin in vitro and in cells, and demonstrated that kindlin is important for normal migfilin dynamics in cells. We also show that when the C-terminal LIM domain region is deleted, then the N-terminal filamin-binding region of the protein, which is capable of targeting migfilin to actin-rich stress fibers, is the predominant driver of migfilin localization. Our work details a correlation between migfilin domains that drive kindlin binding and those that drive FA localization as well as a kindlin dependence on migfilin FA recruitment and mobility. We therefore suggest that the kindlin interaction with migfilin LIM domains drives migfilin FA recruitment, localization, and mobility.     

Wee1 Inhibition by MK-1775 Leads to Tumor Inhibition and Enhances Efficacy of Gemcitabine in Human Sarcomas

Sarcomas are rare and heterogeneous mesenchymal tumors affecting both pediatric and adult populations with more than 70 recognized histologies. Doxorubicin and ifosfamide have been the main course of therapy for treatment of sarcomas; however, the response rate to these therapies is about 10–20% in metastatic setting. Toxicity with the drug combination is high, response rates remain low, and improvement in overall survival, especially in the metastatic disease, remains negligible and new agents are needed. Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 by MK1775 has been reported to enhance the cytotoxic effect of DNA damaging agents in different types of carcinomas. In this study we investigated the therapeutic efficacy of MK1775 in various sarcoma cell lines, patient-derived tumor explants ex vivo and in vivo both alone and in combination with gemcitabine, which is frequently used in the treatment of sarcomas. Our data demonstrate that MK1775 treatment as a single agent at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death in all sarcomas tested. Additionally, MK1775 significantly enhances the cytotoxic effect of gemcitabine in sarcoma cells lines with different p53 mutational status. In patient-derived bone and soft tissue sarcoma samples we showed that MK1775 alone and in combination with gemcitabine causes significant apoptotic cell death. Magnetic resonance imaging (MRI) and histopathologic studies showed that MK1775 induces significant cell death and terminal differentiation in a patient-derived xenograft mouse model of osteosarcoma in vivo. Our results together with the high safety profile of MK1775 strongly suggest that this drug can be used as a potential therapeutic agent in the treatment of both adult as well as pediatric sarcoma patients.     

Facile measurement of protein stability and folding kinetics using a nano differential scanning fluorimeter

With advancements in high‐throughput generation of phenotypic data on mutant proteins, it has become important to individually characterize different proteins or their variants rapidly and with minimal sample consumption. We have made use of a nano differential scanning fluorimetric device, from NanoTemper technologies, to rapidly carry out isothermal chemical denaturation and measure folding/unfolding kinetics of proteins and compared these to corresponding data obtained from conventional spectrofluorimetry. We show that using sample volumes 10‐50‐fold lower than with conventional fluorimetric techniques, one can rapidly and accurately measure thermodynamic and kinetic stability, as well as folding/unfolding kinetics. This method also facilitates characterization of proteins that are difficult to express and purify.     

The structure-specific nicking of small heteroduplexes by the RAG complex: implications for lymphoid chromosomal translocations

During V(D)J recombination, the RAG complex binds at recombination signal sequences and creates double-strand breaks. In addition to this sequence-specific recognition of the RSS, the RAG complex has been shown to be a structure-specific nuclease, cleaving 3' overhangs and 3' flaps, and, more recently, 10 nucleotides (nt) bubble (heteroduplex) structures. Here, we assess the smallest size heteroduplex that core and full-length RAGs can cleave. We also test whether bubbles adjacent to a partial RSS are nicked any differently or any more efficiently than bubbles that are surrounded by random sequence. These points are important in considering what types and what size of non-B DNA structure that the RAG complex can nick, and this helps assess the role of the RAG complex in mediating lymphoid chromosomal translocations. We find that the smallest bubble nicked by the RAG complex is 3nt, and proximity to a partial or full RSS sequence does not affect the nicking by RAGs. RAG nicking efficiency increases with the size of the heteroduplex and is only about two-fold less efficient than an RSS when the bubble is 6nt. We consider these findings in the context of RAG nicking at non-B DNA structures in lymphoid chromosomal translocations.     

Vulnerability of synaptosomes from apoE knock-out mice to structural and oxidative modifications induced by A beta(1-40): implications for Alzheimer's disease

Apolipoprotein E (apoE) plays an important role in the response to central nervous system injury. The e4 allele of apoE and amyloid beta-peptide (Abeta) are associated with Alzheimer's disease (AD) and may be central to the pathogenesis of this disorder. Recent studies demonstrate evidence for neurodegeneration and increased lipid peroxidation in transgenic mice lacking apoE (KO). In the current study, synaptosomes were prepared from apoE KO mice to determine the role of apoE in synaptic membrane structure and to determine susceptibility to oxidative damage by Abeta(1-40). ApoE KO mice exhibited structural modifications to lipid and protein components of synaptosomal membranes as determined by electron paramagnetic resonance in conjunction with lipid- and protein- specific spin labels. Incubation with 5 microM Abeta(1-40) resulted in more severe oxidative modifications to proteins and lipids in apoE KO synaptosomes as measured by protein carbonyls, an index of protein oxidation, and TBARs and protein-bound 4-hydroxynonenal (HNE), markers of lipid oxidation. Together, these data support a role for apoE in the modulation of oxidative injury and in the maintenance of synaptic integrity and are discussed with reference to alterations in AD brain.     

Thermodynamics of replacing an alpha-helical Pro residue in the P40S mutant of Escherichia coli thioredoxin

Escherichia coli thioredoxin is a 108 amino acid oxidoreductase and contains a single Met residue at position 37. The protein contains a long alpha-helical stretch between residues 32 and 49. The central residue of this helix, Pro40, has been replaced by Ser. The stabilities of the oxidized states of two proteins, the single mutant M37L and the double mutant M37L,P40S, have been characterized by differential scanning calorimetry (DSC) and also by a series of isothermal guanidine hydrochloride (GuHCl) melts in the temperature range of 277 to 333 K. The P40S mutation was found to stabilize the protein at all temperatures upto 340 K though both proteins had similar Tm values of about 356 K. At 298 K, the M37L,P40S mutant was found to be more stable than M37L by 1.5 kcal/mol. A combined analysis of GuHCl and calorimetric data was carried out to determine the enthalpy, entropy, and heat capacity change upon unfolding. At 298 K there was a large, stabilizing enthalpic effect in P40S though significant enthalpy-entropy compensation was observed and the two proteins had similar values of deltaCp. Thus, replacement of a Pro in the interior of an alpha helix can have substantial effects on protein stability.     

Elevated oxidative stress in models of normal brain aging and Alzheimer's disease

Age-associated neurodegenerative disorders are becoming more prevalent as the mean age of the population increases in the United States over the next few decades. Both normal brain aging and Alzheimer's disease (AD) are associated with oxidative stress. Our laboratory has used a wide variety of physical and biochemical methods to investigate free radical oxidative stress in several models of aging and AD. Beta-amyloid (A beta), the peptide that constitutes the central core of senile plaques in AD brain, is associated with free radical oxidative stress and is toxic to neurons. This review summarizes some of our studies in aging and A beta-associated free radical oxidative stress and on the modulating effects of free radical scavengers on neocortical synaptosomal membrane damage found in aging and A beta-treated systems.