Mary L. Michaelis

Adenosine modulation of synaptosomal dopamine release.

Abstract The effects of adenosine and its analog 2-chloroadenosine on release of preloaded [ 3 H]-dopamine from striatal synaptosomes was explored. Both adenosine and 2-chloroadenosine were found to decrease the amount of dopamine released both by depolarization (with KCl) and by amphetamine. Addition of exogenous adenosine deaminase enhanced dopamine release above controls, and blockade of the endogenous adenosine deaminase activity with deoxycoformycin resulted in a decrease in dopamine release. The methylxanthines, believed to be adenosine antagonists, inhibited dopamine release by an unknown mechanism, and hence it was impossible to evaluate antagonism of the inhibitory effects of adenosine by these agents. The depolarization-induced release of dopamine appeared to be more sensitive to the actions of adenosine than was the amphetamine-induced release. The data obtained so far seem to indicate that adenosine is capable of modulating the release of transmitter substances in brain tissue in a manner analogous to that which has been observed in the peripheral nervous system.

Age-dependent alterations in synaptic membrane systems for Ca2+ regulation

The effects of aging on two neuronal plasma membrane Ca2+ regulating systems have been examined using synaptic membranes isolated from the brains of adult (5-7-month-old) and aged (23-25-month-old) Fisher 344 rats. The kinetic characteristics of the Na+-dependent Ca2+ transport system were found to be altered in the aged animals. The affinity of the transport carrier for Ca2+ was decreased in membranes from aged animals, with very little change in the maximal transport capacity of the system. The activity of the synaptic membrane Ca2+-activated, Mg2+-dependent ATPase was also altered in membranes from aged animals. In this system, however, the Vmax for Ca2+ activation of the enzymatic activity was lower in the aged animals, while there was no change in the K0.5 for Ca2+ activation. The magnitude of the alterations was small, but the differences were consistent. Even small changes in the effectiveness of the synaptic plasma membrane systems which participate in the maintenance of low intraterminal Ca2+ could progressively affect the integrity of synaptic transmission and lead eventually to neuronal cell death.

High-affinity glutamic acid binding to brain synaptic membranes

Abstract Rat brain homogenate preparations exhibited two types of glutamine binding, one a high-affinity ( K 1 = 0.2 μ M ) and the other a low-affinity type ( K 2 = 4.4 μ M ). The high-affinity binding was primarily associated with the plasma membrane subcellular fractions and in particular with the synaptic membrane subfraction. This l -glutamate binding was found to be strongly stereospecific for the l -form and was almost totally reversible. The synaptic membrane glutamate binding was partialy inhibited by neuro-excitatory and neuro-inhibitory amino acids but was not affected by amino acids lacking in neuropharmacologic activity. The membrane-associated l -glutamate binding system could be solubilized by Triton X-100 without loss of its high-affinity binding activity. The chemical nature of this glutamate binding component was found to be that of a glycolipoprotein. It is proposed that this glutamate binding system represents the physiologic receptor on neuronal membranes of this amino acid.

Decreased plasma membrane calcium transport activity in aging brain

We have assessed the functional properties of both calmodulin (CaM) and the plasma membrane Ca(2+)-ATPase in brains of young, middle aged, and old Fisher 344 rats. Under optimal conditions of saturating Ca2+ and ATP, the CaM-activated Ca(2+)-ATPase activity was decreased with increasing age, particularly when CaM isolated from the brains of aged rats was used to stimulate the enzyme. In the case of CaM, structural modifications within the primary sequence of the protein from aged brains were identified. We found that during normal biological aging approximately 6 methionine residues were modified to their corresonding sulfoxide per CaM, and no other amino acids were modified. Some aspects of the age-related decline in the effectiveness of CaM as an activator of Ca(2+)-ATPase could be simulated using a range of reactive oxygen species (including hydrogen peroxide and oxoperoxynitrite) and, in the latter case, the extent of oxidative modification of specific methionine residues was directly related to their surface accessibility. The pattern of oxidative modification of the methionines in the aged CaM was less straightforward, though both in vitro oxidation of CaM and aging within the brain markedly decreased the functional properties of this important Ca(2+)-regulating protein.

β-Amyloid-Induced Neurodegeneration and Protection by Structurally Diverse Microtubule-Stabilizing Agents

Deposition of β-amyloid peptide (Aβ) and hyperphosphorylation of the τ protein are associated with neuronal dysfunction and cell death in Alzheimers disease. Although the relationship between these two processes is not yet understood, studies have shown that both in vitro and in vivo exposure of neurons to Aβ leads to τ hyperphosphorylation and neuronal dystrophy. We previously reported that the microtubule-stabilizing drug paclitaxel (Taxol) protects primary neurons against toxicity induced by the Aβ25-35 peptide. The studies in this report were undertaken to characterize the actions of paclitaxel more fully, to assess the effectiveness of structurally diverse microtubulestabilizing agents in protecting neurons, and to determine the time course of the protective effects of the drugs. Primary neurons were exposed to Aβ in the presence or absence of several agents shown to interact with microtubules, and neuronal survival was monitored. Paclitaxel protected neurons against Aβ1-42 toxicity, and paclitaxel-treated cultures exposed to Aβ showed enhanced survival over Aβ-only cultures for several days. Neuronal apoptosis induced by Aβ was blocked by paclitaxel. Other taxanes and three structurally diverse microtubule-stabilizing compounds also significantly increased survival of Aβ-treated cultures. At concentrations below 100 nM, the drugs that protected the neurons did not produce detectable toxicity when added to the cultures alone. Although multiple mechanisms are likely to contribute to the neuronal cell death induced by oligomeric or fibrillar forms of Aβ, low concentrations of drugs that preserve the integrity of the cytoskeletal network may help neurons survive the toxic cascades initiated by these peptides.

Protection Against β‐Amyloid Toxicity in Primary Neurons by Paclitaxel (Taxol)

Abstract: Neurofibrillary tangles in Alzheimers disease contain aggregates of abnormally phosphorylated microtubule‐associated protein τ, indicating that microtubule breakdown is a primary event in the neurodegenerative cascade. Recent studies have shown that addition to neuronal cultures of amyloid peptides found in Alzheimers leads to abnormal phosphorylation of τ and neurofibrillary pathology. We tested the possibility that the microtubule‐stabilizing drug paclitaxel (Taxol) might protect primary neurons against amyloid‐induced toxicity. Neurons exposed to aggregated amyloid peptides 25–35 and 1–42 became pyknotic with degenerating neurites within 24 h. Treatment of cultures with paclitaxel either 2 h before or 2 h after addition of the peptide prevented these morphological alterations. When numbers of viable cells were determined in cultures exposed to amyloid peptide with or without paclitaxel for 24 or 96 h, the percentage of surviving cells was significantly higher in paclitaxel‐treated cultures, and activation of the apoptosis‐associated protease CPP32 was significantly reduced. These observations indicate that microtubule‐stabilizing drugs may help slow development of the neurofibrillary pathology that leads to the loss of neuronal integrity in Alzheimers disease.

Age-related decrease in brain synaptic membrane Ca2+-ATPase in F344/BNF1 rats.

We used Fisher 344/Brown Norway hybrid rats (F344/BNF1) to determine whether previously reported decreases in brain synaptic plasma membrane (SPM) Ca2+-ATPase activity in inbred F344 rats also occurred in the hybrids. Plasma membrane Ca2+-ATPase (PMCA) activity in SPMs from F344/BNF1 rat brains showed a progressive age-dependent decrease in Vmax from 60.9 +/- 3.7 nmol Pi/mg/min (n = 6) in 5-month rats to 32.4 +/- 3.6 nmol Pi/mg/min (n = 6) in 34-month animals, with no change in K (act) for Ca2+. Immunoreactive PMCA in SPMs also decreased by approximately 20% at 34 months, and the calmodulin (CaM) bound to membranes following extraction with EDTA also declined progressively with age. The effectiveness of CaM in stimulating PMCA activity was significantly lower when CaM was purified from the brains of old vs. young F344 rats and when CaM from 5-month rats was oxidized in vitro. These results indicate: 1) that PMCA activity in SPMs from longer lived F344/BNF1 hybrids also decreases with age; 2) that part of the reduction in PMCA activity is due to loss of PMCA from the membranes; and 3) that age-related structural changes in CaM may decrease its interaction with proteins in SPMs.

Regulation of calcium levels in brain tissue from adult and aged rats.

The possibility that regulation of Ca2+ levels in brain nerve terminals is altered as the brain ages was examined in synaptosomes from adult and aged Fischer 344 rats. Free intrasynaptosomal [Ca2+]i was monitored with fura-2 as synaptosomes were depolarized with KCl, veratridine and ibotenic acid. With all three depolarizing agents, synaptosomes from aged animals reached higher free Ca2+ levels, and the maximal Ca2+ increases (delta Ca2+) estimated from computer assisted-fitting of the curves, ranged from 35% to 80% greater in synaptosomes from aged animals. The total Ca2+ content of the brain and of synaptosomes was also found to be considerably higher in aged than in adult animals. These results suggest that the aging process in brain is accompanied by alterations in both dynamic aspects of Ca2+ handling in nerve endings and the overall content of Ca2+ in the brain and synaptic terminals.

Stabilization of the cyclin‐dependent kinase 5 activator, p35, by paclitaxel decreases β‐amyloid toxicity in cortical neurons

One hallmark of Alzheimers disease (AD) is the formation of neurofibrillary tangles, aggregated paired helical filaments composed of hyperphosphorylated tau. Amyloid‐β (Aβ) induces tau hyperphosphorylation, decreases microtubule (MT) stability and induces neuronal death. MT stabilizing agents have been proposed as potential therapeutics that may minimize Aβ toxicity and here we report that paclitaxel (taxol) prevents cell death induced by Aβ peptides, inhibits Aβ‐induced activation of cyclin‐dependent kinase 5 (cdk5) and decreases tau hyperphosphorylation. Taxol did not inhibit cdk5 directly but significantly blocked Aβ‐induced calpain activation and decreased formation of the cdk5 activator, p25, from p35. Taxol specifically inhibited the Aβ‐induced activation of the cytosolic cdk5‐p25 complex, but not the membrane‐associated cdk5‐p35 complex. MT‐stabilization was necessary for neuroprotection and inhibition of cdk5 but was not sufficient to prevent cell death induced by overexpression of p25. As taxol is not permeable to the blood–brain barrier, we assessed the potential of taxanes to attenuate Aβ toxicity in adult animals using a succinylated taxol analog (TX67) permeable to the blood–brain barrier. TX67, but not taxol, attenuated the magnitude of both basal and Aβ‐induced cdk5 activation in acutely dissociated cortical cultures prepared from drug treated adult mice. These results suggest that MT‐stabilizing agents may provide a therapeutic approach to decrease Aβ toxicity and neurofibrillary pathology in AD and other tauopathies.

Transgenic Expression of Glud1 (Glutamate Dehydrogenase 1) in Neurons: In Vivo Model of Enhanced Glutamate Release, Altered Synaptic Plasticity, and Selective Neuronal Vulnerability

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.