Mary L Michaelis

University of Kansas, Lawrence, Kansas, United States

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Publications (46)138.92 Total impact

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    ABSTRACT: Control of intracellular calcium concentrations ([Ca2 +]i) is essential for neuronal function, and the plasma membrane Ca2 +-ATPase (PMCA) is crucial for the maintenance of low [Ca2 +]i. We previously reported on loss of PMCA activity in brain synaptic membranes during aging. Gangliosides are known to modulate Ca2 + homeostasis and signal transduction in neurons. In the present study, we observed age-related changes in the ganglioside composition of synaptic plasma membranes. This led us to hypothesize that alterations in ganglioside species might contribute to the age-associated loss of PMCA activity. To probe the relationship between changes in endogenous ganglioside content or composition and PMCA activity in membranes of cortical neurons, we induced depletion of gangliosides by treating neurons with d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (d-PDMP). This caused a marked decrease in the activity of PMCA, which suggested a direct correlation between ganglioside content and PMCA activity. Neurons treated with neuraminidase exhibited an increase in GM1 content, a loss in poly-sialoganglioside content, and a decrease in PMCA activity that was greater than that produced by d-PDMP treatment. Thus, it appeared that poly-sialogangliosides had a stimulatory effect whereas mono-sialogangliosides had the opposite effect. Our observations add support to previous reports of PMCA regulation by gangliosides by demonstrating that manipulations of endogenous ganglioside content and species affect the activity of PMCA in neuronal membranes. Furthermore, our studies suggest that age-associated loss in PMCA activity may result in part from changes in the lipid environment of this Ca2+ transporter.
    Biochimica et Biophysica Acta (BBA) - Biomembranes 05/2014; 1838(5):1255-1265. · 3.39 Impact Factor
  • Biochimica et Biophysica Acta (BBA) - Biomembranes. 01/2014; 1838(5):1255–1265.
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    ABSTRACT: Precise regulation of free intracellular Ca(2+) concentrations [Ca(2+) ](i) is critical for normal neuronal function, and alterations in Ca(2+) homeostasis are associated with brain aging and neurodegenerative diseases. One of the most important proteins controlling [Ca(2+) ](i) is the plasma membrane Ca(2+) -ATPase (PMCA), the high-affinity transporter that fine tunes the cytosolic nanomolar levels of Ca(2+) . We previously found that PMCA protein in synaptic plasma membranes (SPMs) is decreased with advancing age and the decrease in enzyme activity is much greater than that in protein levels. In this study, we isolated raft and non-raft fractions from rat brain SPMs and used quantitative mass spectrometry to show that the specialized lipid microdomains in SPMs, the rafts, contain 60% of total PMCA, comprised all four isoforms. The raft PMCA pool had the highest specific activity and this decreased progressively with age. The reduction in PMCA protein could not account for the dramatic activity loss. Addition of excess calmodulin to the assay did not restore PMCA activity to that in young brains. Analysis of the major raft lipids revealed a slight age-related increase in cholesterol levels and such increases might enhance membrane lipid order and prevent further loss of PMCA activity.
    Journal of Neurochemistry 08/2012; · 3.97 Impact Factor
  • Huiping Zhao, Mary L Michaelis, Brian S J Blagg
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    ABSTRACT: Hsp90 serves as the master regulator of the prosurvival, heat shock response. Upon exposure to cellular stress or small molecule inhibitors of Hsp90, various heat shock proteins are induced to assist in the rematuration of misfolded proteins. Several neurodegenerative diseases, including Alzheimer's disease, manifest through the accumulation of misfolded proteins, suggesting that induction of the heat shock response may provide a viable approach toward the management of such diseases. In this chapter, the rationale for such an approach and potential therapeutics are discussed.
    Advances in pharmacology (San Diego, Calif.) 01/2012; 64:1-25.
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    ABSTRACT: We have previously shown that overexpression of the Glud1 (glutamate dehydrogenase 1) gene in neurons of C57BL/6 mice results in increased depolarization-induced glutamate release that eventually leads to selective neuronal injury and cell loss by 12 months of age. However, it is known that isogenic lines of Tg (transgenic) mice produced through back-crossing with one strain may differ in their phenotypic characteristics from those produced using another inbred mouse strain. Therefore, we decided to introduce the Glud1 transgene into the Balb/c strain that has endogenously lower levels of GLUD1 (glutamate dehydrogenase 1) enzyme activity in the brain as compared with C57BL/6. Using an enzyme-based MEA (microelectrode array) that is selective for measuring glutamate in vivo, we measured depolarization-induced glutamate release. Within a discrete layer of the striatum, glutamate release was significantly increased in Balb/c Tg mice compared with wt (wild-type) littermates. Furthermore, Balb/c mice released approx. 50-60% of the amount of glutamate compared with C57BL/6 mice. This is similar to the lower levels of endogenous GLUD1 protein in Balb/c compared with C57BL/6 mice. The development of these Glud1-overexpressing mice may allow for the exploration of key molecular events produced by chronic exposure of neurons to moderate, transient increases in glutamate release, a process hypothesized to occur in neurodegenerative disorders.
    03/2011; 3(2). · 3.64 Impact Factor
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    Jieun Kim, In-Young Choi, Mary L Michaelis, Phil Lee
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    ABSTRACT: Impaired axonal transport has been linked to the pathogenic processes of Alzheimer's disease (AD) in which axonal swelling and degeneration are prevalent. The development of non-invasive neuroimaging methods to quantitatively assess in vivo axonal transport deficits would be enormously valuable to visualize early, yet subtle, changes in the AD brain, to monitor the disease progression and to quantify the effect of drug intervention. A triple transgenic mouse model of AD closely resembles human AD neuropathology. In this study, we investigated age-dependent alterations of the axonal transport rate in the triple transgenic mouse olfactory system, using fast multi-sliced T(1) mapping with manganese-enhanced MRI. The data show that impairment in axonal transport is a very early event in AD pathology in these mice, preceding both deposition of Aβ plaques and formation of Tau fibrils.
    NeuroImage 02/2011; 56(3):1286-92. · 6.25 Impact Factor
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    Xinkun Wang, Mary L Michaelis, Elias K Michaelis
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    ABSTRACT: Pivotal brain functions, such as neurotransmission, cognition, and memory, decline with advancing age and, especially, in neurodegenerative conditions associated with aging, such as Alzheimer's disease (AD). Yet, deterioration in structure and function of the nervous system during aging or in AD is not uniform throughout the brain. Selective neuronal vulnerability (SNV) is a general but sometimes overlooked characteristic of brain aging and AD. There is little known at the molecular level to account for the phenomenon of SNV. Functional genomic analyses, through unbiased whole genome expression studies, could lead to new insights into a complex process such as SNV. Genomic data generated using both human brain tissue and brains from animal models of aging and AD were analyzed in this review. Convergent trends that have emerged from these data sets were considered in identifying possible molecular and cellular pathways involved in SNV. It appears that during normal brain aging and in AD, neurons vulnerable to injury or cell death are characterized by significant decreases in the expression of genes related to mitochondrial metabolism and energy production. In AD, vulnerable neurons also exhibit down-regulation of genes related to synaptic neurotransmission and vesicular transport, cytoskeletal structure and function, and neurotrophic factor activity. A prominent category of genes that are up-regulated in AD are those related to inflammatory response and some components of calcium signaling. These genomic differences between sensitive and resistant neurons can now be used to explore the molecular underpinnings of previously suggested mechanisms of cell injury in aging and AD.
    Current Genomics 12/2010; 11(8):618-33. · 2.48 Impact Factor
  • Jieun Kim, In-Young Choi, Mary L. Michaelis, Sang-Pil Lee
    Alzheimers & Dementia - ALZHEIMERS DEMENT. 01/2010; 6(4).
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    ABSTRACT: The effects of lifelong, moderate excess release of glutamate (Glu) in the CNS have not been previously characterized. We created a transgenic (Tg) mouse model of lifelong excess synaptic Glu release in the CNS by introducing the gene for glutamate dehydrogenase 1 (Glud1) under the control of the neuron-specific enolase promoter. Glud1 is, potentially, an important enzyme in the pathway of Glu synthesis in nerve terminals. Increased levels of GLUD protein and activity in CNS neurons of hemizygous Tg mice were associated with increases in the in vivo release of Glu after neuronal depolarization in striatum and in the frequency and amplitude of miniature EPSCs in the CA1 region of the hippocampus. Despite overexpression of Glud1 in all neurons of the CNS, the Tg mice suffered neuronal losses in select brain regions (e.g., the CA1 but not the CA3 region). In vulnerable regions, Tg mice had decreases in MAP2A labeling of dendrites and in synaptophysin labeling of presynaptic terminals; the decreases in neuronal numbers and dendrite and presynaptic terminal labeling increased with advancing age. In addition, the Tg mice exhibited decreases in long-term potentiation of synaptic activity and in spine density in dendrites of CA1 neurons. Behaviorally, the Tg mice were significantly more resistant than wild-type mice to induction and duration of anesthesia produced by anesthetics that suppress Glu neurotransmission. The Glud1 mouse might be a useful model for the effects of lifelong excess synaptic Glu release on CNS neurons and for age-associated neurodegenerative processes.
    Journal of Neuroscience 11/2009; 29(44):13929-44. · 6.91 Impact Factor
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    ABSTRACT: Compounds that interact with microtubules, such as paclitaxel, have been shown to possess protective properties against beta-amyloid (Abeta) induced neurodegeneration associated with Alzheimer's disease. In this work, the novel agent (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol was investigated for effectiveness in protecting neurons against several toxic stimuli and its interaction with the microtubule network. Exposure of neuronal cultures to Abeta peptide in the presence of 5 nM (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol resulted in a 50% increase in survival. Neuronal cultures treated with other toxic stimuli such as staurosporine, thapsigargin, paraquat, and H(2)O(2) showed significantly enhanced survival in the presence of (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol. Microtubule binding and tubulin assembly studies revealed differences compared to paclitaxel but confirmed the interaction of (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol with microtubules. Furthermore, in vitro studies using bovine brain microvessel endothelial cells experiments suggest that (3R,5S,7as)-(3,5-bis(4-fluorophenyl)tetrahydro-1H-oxazolo[3,4-c]oxazol-7a-yl)methanol can readily cross the blood-brain barrier in a passive manner.
    Journal of Medicinal Chemistry 10/2009; 52(23):7537-43. · 5.61 Impact Factor
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    ABSTRACT: A variety of approaches have been taken to improve the brain penetration of pharmaceutical agents. The amphipathic character of a compound can improve its interaction with the lipid bilayer within cell membranes, and as a result improve permeability. Fatty acid chains or lipoamino acids of various lengths were attached to tranylcypromine (TCP), in an attempt to improve the blood-brain barrier (BBB) permeability by increasing the lipophilicity as well as the amphiphatic character of the drug. TCP-FA4, one of the derivatives containing a four carbon alkyl acid chain, showed the greatest improvement in permeability. This molecule was slightly neuroprotective in a beta-amyloid-induced neurodegeneration assay and may also be capable of upregulating brain derived neurotrophic factor (BDNF), as indicated by cell culture assays using human umbilical vein endothelial cells. Since decreased levels of BDNF are observed in many CNS disorders, and direct injection of BDNF is not a viable option due to its poor permeability across the BBB, small molecules capable of regulating BDNF that also cross the BBB may be an interesting treatment option.
    Biochemical pharmacology 09/2009; 78(11):1412-7. · 4.25 Impact Factor
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    ABSTRACT: Oxidative stress (OS) is an important factor in brain aging and neurodegenerative diseases. Certain neurons in different brain regions exhibit selective vulnerability to OS. Currently little is known about the underlying mechanisms of this selective neuronal vulnerability. The purpose of this study was to identify endogenous factors that predispose vulnerable neurons to OS by employing genomic and biochemical approaches. In this report, using in vitro neuronal cultures, ex vivo organotypic brain slice cultures and acute brain slice preparations, we established that cerebellar granule (CbG) and hippocampal CA1 neurons were significantly more sensitive to OS (induced by paraquat) than cerebral cortical and hippocampal CA3 neurons. To probe for intrinsic differences between in vivo vulnerable (CA1 and CbG) and resistant (CA3 and cerebral cortex) neurons under basal conditions, these neurons were collected by laser capture microdissection from freshly excised brain sections (no OS treatment), and then subjected to oligonucleotide microarray analysis. GeneChip-based transcriptomic analyses revealed that vulnerable neurons had higher expression of genes related to stress and immune response, and lower expression of energy generation and signal transduction genes in comparison with resistant neurons. Subsequent targeted biochemical analyses confirmed the lower energy levels (in the form of ATP) in primary CbG neurons compared with cortical neurons. Low energy reserves and high intrinsic stress levels are two underlying factors for neuronal selective vulnerability to OS. These mechanisms can be targeted in the future for the protection of vulnerable neurons.
    BMC Neuroscience 03/2009; 10:12. · 3.00 Impact Factor
  • Sang-Pil Lee, Jieun Kim, Mary L. Michaelis, In-Young Choi
    Alzheimers & Dementia - ALZHEIMERS DEMENT. 01/2009; 5(4).
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    ABSTRACT: Brain aging is associated with a progressive decline in cognitive function though the molecular mechanisms remain unknown. Functional changes in brain neurons could be due to age-related alterations in levels of specific proteins critical for information processing. Specialized membrane microdomains known as 'lipid rafts' contain protein complexes involved in many signal transduction processes. This study was undertaken to determine if two-dimensional fluorescence difference gel electrophoresis (2D DIGE) analysis of proteins in synaptic membrane lipid rafts revealed age-dependent alterations in levels of raft proteins. Five pairs of young and aged rat synaptic membrane rafts were subjected to DIGE separation, followed by image analysis and identification of significantly altered proteins. Of 1046 matched spots on DIGE gels, 94 showed statistically significant differences in levels between old and young rafts, and 87 of these were decreased in aged rafts. The 41 most significantly altered (p<0.03) proteins included several synaptic proteins involved in energy metabolism, redox homeostasis, and cytoskeletal structure. This may indicate a disruption in bioenergetic balance and redox homeostasis in synaptic rafts with brain aging. Differential levels of representative identified proteins were confirmed by immunoblot analysis. Our findings provide novel pathways in investigations of mechanisms that may contribute to altered neuronal function in aging brain.
    Neurobiology of aging 01/2009; 31(12):2146-59. · 5.94 Impact Factor
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    ABSTRACT: Alzheimer's disease (AD) neuropathology is characterized by loss of synapses and neurons, neuritic plaques consisting of beta-amyloid (Abeta) peptides, and neurofibrillary tangles consisting of intracellular aggregates of hyperphosphorylated tau protein in susceptible brain regions. Abeta oligomers trigger a cascade of pathogenic events including tau hyperphosphorylation and aggregation, inflammatory reactions, and excitotoxicity that contribute to the progression of AD. The molecular chaperone Hsp90 facilitates the folding of newly synthesized and denatured proteins and is believed to play a role in neurodegenerative disorders in which the defining pathology results in misfolded proteins and the accumulation of protein aggregates. Some agents that inhibit Hsp90 protect neurons against Abeta toxicity and tau aggregation, and assays for rapidly screening potential Hsp90 inhibitors are of interest. We used the release of the soluble cytosolic enzyme lactate dehydrogenase (LDH) as an indicator of the loss of cell membrane integrity and cytotoxicity resulting from exposure to Abeta peptides to evaluate the neuroprotective properties of novel novobiocin analogues and established Hsp90 inhibitors. Compounds were assessed for potency in protecting proliferating and differentiated SH-SY5Y neuronal cells against Abeta-induced cell death; the potential toxicity of each agent alone was also determined. The data indicated that several of the compounds decreased Abeta toxicity even at low nanomolar concentrations and, unexpectedly, were more potent in protecting the undifferentiated cells against Abeta. The novobiocin analogues alone were not toxic even up to 10 microM concentrations whereas GDA and the parent compound, novobiocin, were toxic at 1 and 10 microM, respectively. The results suggest that novobiocin analogues may provide novel leads for the development of neuroprotective drugs.
    Bioorganic & medicinal chemistry 01/2009; 17(4):1709-15. · 2.82 Impact Factor
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    ABSTRACT: Oxidative stress leads to the disruption of calcium homeostasis in brain neurons; however, the direct effects of oxidants on proteins that regulate intracellular calcium ([Ca2+]i) are not known. The calmodulin (CaM)-stimulated plasma membrane Ca2+-ATPase (PMCA) plays a critical role in regulating [Ca2+]i. Our previous in vitro studies showed that PMCA present in brain synaptic membranes is readily inactivated by a variety of reactive oxygen species (ROS). The present studies were conducted to determine the vulnerability of PMCA to ROS generated in neurons as would probably occur in vivo. Primary cortical neurons were exposed to paraquat (PQ), a redox cycling agent that generates intracellular ROS. Low concentrations of PQ (5–10 μM) increased PMCA basal activity by two-fold but abolished its sensitivity to CaM. Higher concentrations (25–100 μM) inhibited both components of PMCA activity. Immunoblots showed the formation of high-molecular-weight PMCA aggregates. Additionally, PMCA showed evidence of proteolytic degradation. PMCA proteolysis was prevented by a calpain inhibitor, suggesting a role for calpain. Our findings suggest that PMCA is a sensitive target of oxidative stress in primary neurons. Inactivation of this Ca2+ transporter under prolonged oxidative stress could alter neuronal Ca2+ signaling.
    Free Radical Biology and Medicine. 01/2009;
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    ABSTRACT: Calpain activation is hypothesized to be an early occurrence in the sequence of events resulting in neurodegeneration, as well as in the signaling pathways linking extracellular accumulation of beta-amyloid (Abeta) peptides and intracellular formation of neurofibrillary tangles. In an effort to identify small molecules that prevent neurodegeneration in Alzheimer's disease by early intervention in the cell death cascade, a cell-based assay in differentiated Sh-SY5Y cells was developed using calpain activity as a read-out for the early stages of death in cells exposed to extracellular Abeta. This assay was optimized for high-throughput screening, and a library of approximately 120,000 compounds was tested. It was expected that the compounds identified as calpain inhibitors would include those that act directly on the enzyme and those that prevented calpain activation by blocking an upstream step in the pathway. In fact, of the compounds that inhibited calpain activation by Abeta with IC(50) values of <10 microM and showed little or no toxicity at concentrations up to 30 microM, none inhibit the calpain enzyme directly.
    Journal of Biomolecular Screening 09/2008; 13(9):870-8. · 2.21 Impact Factor
  • Alzheimers & Dementia - ALZHEIMERS DEMENT. 01/2008; 4(4).
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    ABSTRACT: Intraneuronal calcium ([Ca(2+)](i)) regulation is altered in aging brain, possibly because of the changes in critical Ca(2+) transporters. We previously reported that the levels of the plasma membrane Ca(2+)-ATPase (PMCA) and the V(max) for enzyme activity are significantly reduced in synaptic membranes in aging rat brain. The goal of these studies was to use RNA(i) techniques to suppress expression of a major neuronal isoform, PMCA2, in neurons in culture to determine the potential functional consequences of a decrease in PMCA activity. Embryonic rat brain neurons and SH-SY5Y neuroblastoma cells were transfected with in vitro--transcribed short interfering RNA or a short hairpin RNA expressing vector, respectively, leading to 80% suppression of PMCA2 expression within 48 h. Fluorescence ratio imaging of free [Ca(2+)](i) revealed that primary neurons with reduced PMCA2 expression had higher basal [Ca(2+)](i), slower recovery from KCl-induced Ca(2+) transients, and incomplete return to pre-stimulation Ca(2+) levels. Primary neurons and SH-SY5Y cells with PMCA2 suppression both exhibited significantly greater vulnerability to the toxicity of various stresses. Our results indicate that a loss of PMCA such as occurs in aging brain likely leads to subtle disruptions in normal Ca(2+) signaling and enhanced susceptibility to stresses that can alter the regulation of Ca(2+) homeostasis.
    Journal of Neurochemistry 08/2007; 102(2):454-65. · 3.97 Impact Factor
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    ABSTRACT: Spatial and temporal alterations in intracellular calcium [Ca(2+)](i) play a pivotal role in a wide array of neuronal functions. Disruption in Ca(2+) homeostasis has been implicated in the decline in neuronal function in brain aging and in neurodegenerative disorders. The plasma membrane Ca(2+)-ATPase (PMCA) is a high affinity Ca(2+) transporter that plays a crucial role in the termination of [Ca(2+)](i) signals and in the maintenance of low [Ca(2+)](i) essential for signaling. Recent evidence indicates that PMCA is uniquely sensitive to its lipid environment and is stimulated by lipids with ordered acyl chains. Here we show that both PMCA and its activator calmodulin (CaM) are partitioned into liquid-ordered, cholesterol-rich plasma membrane microdomains or 'lipid rafts' in primary cultured neurons. Association of PMCA with rafts was demonstrated in preparations isolated by sucrose density gradient centrifugation and in intact neurons by confocal microscopy. Total raft-associated PMCA activity was much higher than the PMCA activity excluded from these microdomains. Depletion of cellular cholesterol dramatically inhibited the activity of the raft-associated PMCA with no effect on the activity of the non-raft pool. We propose that association of PMCA with rafts represents a novel mechanism for its regulation and, consequently, of Ca(2+) signaling in the central nervous system.
    Journal of Neurochemistry 08/2007; 102(2):378-88. · 3.97 Impact Factor