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The molecular-level polymorphism in ß-Amyloid (Aß) fibrils have recently been considered as a pathologically relevant factor in Alzheimer's disease (AD). Studies showed that the structural deviations in human-brain-seeded Aß fibrils potentially correlated with the clinical histories of AD patients. For the 40-residue Aß (Aß40) fibrils derived from human brain tissues, a predominant molecular structure was proposed based on solid-state nuclear magnetic resonance (ssNMR) spectroscopy. However, previous studies have shown that the molecular structures of Aß 40 fibrils were sensitive to their growth conditions in aqueous environments. We show in this work that biological membranes and their phospholipid bilayer mimics serve as environmental factors to reduce the structural heterogeneity in Aß40 fibrils. Fibrillization in the presence of membranes leads to fibril structures that are significantly different to the Aß40 fibrils grown in aqueous solutions. Fibrils grown from multiple types of membranes, including the biological membranes extracted from the rats' synaptosomes, shared similar ssNMR spectral features. Our studies emphasize the biological relevance of membranes in Aß40 fibril structures and fibrillization processes.

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Energy metabolism is fundamental to maintain Central Nervous System homeostasis because of high requirement of adenosine triphosphate (ATP), that is necessary to sustain neuronal events. During aging, changes in brain bioenergetics may influence the recovery of cerebral tissue in coping with pathophysiological conditions and pharmacological treatments. For this reason, we have previously studied enzyme catalytic activities related to energy-yielding systems. In the present study, the maximum rates (Vmax) of some enzymatic activities related to energy consumption (ATPases) were evaluated on synaptic plasma membranes (S.P.M.) isolated from frontal cerebral cortex of male Wistar rats aged 2, 6, 12, 18 and 24 months, because of the key role of these enzymes in modulating presynaptic nerve ending homeostasis. The following enzyme activities were evaluated: Na+, K+, Mg2+-ATPase; ouabain-insensitive Mg2+-ATPase; Na+, K+-ATPase; specific Mg2+-ATPase; Ca2+, Mg2+-ATPase; acetylcholinesterase (AChE). The present results show that both the activities of (i) ATPases and (ii) AChE were significantly decreased during aging. Comparing these observations with those previously done on rat striatum on the same functional parameters and in the same experimental settings, ATPases activities were influenced by the age factor in different ways, suggesting that the frontal cerebral cortex independently adapt to the different age-dependent biochemical situations at each single age. Overall, this experimental approach is therefore important to add pieces of information for the understanding of the correlation between aging and brain energy metabolism, and could be a suitable model to assess also drug effects, differentiating between different cerebral areas.

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Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches. For example, structural features of the synaptic terminals and the organelles within them, such as synaptic vesicles and mitochondria, have been elucidated with electron microscopy. The postsynaptic membranes are joined to the presynaptic "active zone" of transmitter release through cell adhesion molecules and remain attached throughout the isolation of synaptosomes. These "post synaptic densities" or "PSDs" contain the receptors for the transmitters released from the nerve terminals and can easily be seen with electron microscopy. Biochemical and cell biological studies with synaptosomes have revealed which proteins and lipids are most actively involved in synaptic release of neurotransmitters. The functional properties of the nerve terminals, such as responses to depolarization and the uptake or release of signaling molecules, have also been characterized through the use of fluorescent dyes, tagged transmitters, and transporter substrates. In addition, isolated synaptosomes can serve as the starting material for the isolation of relatively pure synaptic plasma membranes (SPMs) that are devoid of organelles from the internal environment of the nerve terminal, such as mitochondria and synaptic vesicles. The isolated SPMs can reseal and form vesicular structures in which transport of ions such as sodium and calcium, as well as solutes such as neurotransmitters can be studied. The PSDs also remain associated with the presynaptic membranes during isolation of SPM fractions, making it possible to isolate the synaptic junctional complexes (SJCs) devoid of the rest of the plasma membranes of the nerve terminals and postsynaptic membrane components. Isolated SJCs can be used to identify the proteins that constitute this highly specialized region of neurons. In this chapter, we describe the steps involved in isolating synaptosomes, SPMs, and SJCs from brain so that these preparations can be used with new technological advances to address many as yet unanswered questions about the synapse and its remarkable activities in neuronal cell communication.

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The maximum rate (Vmax) of some enzymatic activities related to energy consumption was evaluated in synaptic plasma membranes from rat brain striatum, the synaptic energy state being a crucial factor in neurodegenerative diseases etiopathogenesis. Two types of synaptic plasma membranes were isolated from rats subjected to in vivo treatment with L-acetylcarnitine at two different doses (30 and 60 mg × kg(-1) i.p., 28 days, 5 days/week). The following enzyme activities were evaluated: acetylcholinesterase (AChE); Na(+), K(+), Mg(2+)-ATP-ase; ouabain insensitive Mg(2+)-ATP-ase; Na(+), K(+)-ATP-ase; direct Mg(2+)-ATP-ase; Ca(2+), Mg(2+)-ATP-ase; and low- and high-affinity Ca(2+)-ATP-ase. In control (vehicle-treated) animals, enzymatic activities are differently expressed in synaptic plasma membranes type I (SPM1) with respect to synaptic plasma membranes type II (SPM2), the evaluated enzymatic activities being higher in SPM2. Subchronic treatment with L-acetylcarnitine decreased AChE on SPM1 and SPM2 at the dose of 30 mg × kg(-1). Pharmacological treatment decreased ouabain insensitive Mg(2+)-ATP-ase activity and high affinity Ca(2+)-ATP-ase activity at the doses of 30 and 60 mg × kg(-1) respectively on SPM1, while it decreased Na(+), K(+)-ATP-ase, direct Mg(2+)-ATP-ase and Ca(2+), Mg(2+)-ATP-ase activities at the dose of 30 mg × kg(-1) on SPM2. These results suggest that the sensitivity to drug treatment is different between these two populations of synaptic plasma membranes from the striatum, confirming the micro-heterogeneity of these subfractions, possessing different metabolic machinery with respect to energy consumption and utilization and the regional selective effect of L-acetylcarnitine on cerebral tissue, depending on the considered area. The drug potential effect at the synaptic level in Parkinson's Disease neuroprotection is also discussed with respect to acetylcholine and energy metabolism.

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The maximum rates of adenosine triphosphatase (ATPase) systems related to energy consumption were systematically evaluated in synaptic plasma membranes isolated from the striata of male Wistar rats aged 2, 6, 12, 18, and 24 months, because of their key role in presynaptic nerve ending homeostasis. The following enzyme activities were evaluated: sodium-potassium-magnesium adenosine triphosphatase (Na(+), K(+), Mg(2+)-ATPase); ouabain-insensitive magnesium adenosine triphosphatase (Mg(2+)-ATPase); sodium-potassium adenosine triphosphatase (Na(+), K(+)-ATPase); direct magnesium adenosine triphosphatase (Mg(2+)-ATPase); calcium-magnesium adenosine triphosphatase (Ca(2+), Mg(2+)-ATPase); and acetylcholinesterase. The results showed that Na(+), K(+)-ATPase decreased at 18 and 24 months, Ca(2+), Mg(2+)-ATPase and acetylcholinesterase decreased from 6 months, while Mg(2+)-ATPase was unmodified. Therefore, ATPases vary independently during aging, suggesting that the ATPase enzyme systems are of neuropathological and pharmacological importance. This could be considered as an experimental model to study regeneration processes, because of the age-dependent modifications of specific synaptic plasma membranes. ATPases cause selective changes in some cerebral functions, especially bioenergetic systems. This could be of physiopathological significance, particularly in many central nervous system diseases, where, during regenerative processes, energy availability is essential.