[11C]physostigmine, an acetylcholinesterase inhibitor, has been shown to be a promising positron emission tomography ligand to quantify the cerebral concentration of the enzyme in animals and humans in vivo. Here, a quantitative and noninvasive method to measure the regional acetylcholinesterase concentration in the brain is presented. The method is based on the observation that the ratio between regions rich in acetylcholinesterase and white matter, a region almost entirely deprived of this enzyme, was found to become approximately constant after 20 to 30 minutes, suggesting that at late time points the uptake mainly contains information about the distribution volume. Taking the white matter as the reference region, a simplified reference tissue model, with effectively one reversible tissue compartment and three parameters, was found to give a good description of the data in baboons. One of these parameters, the ratio between the total distribution volumes in the target and reference regions, showed a satisfactory correlation with the acetylcholinesterase concentration measured postmortem in two baboon brains. Eight healthy male subjects were also analyzed and the regional enzyme concentrations obtained again showed a good correlation with the known acetylcholinesterase concentrations measured in postmortem studies of human brain.
It is unclear whether the palliative effects of tetrahydroaminoacridine (THA) (tacrine, Cognex) on the clinical symptoms of patients affected by Alzheimer's disease (AD) are the result of its inhibitory activity on acetylcholinesterase or on other complex sites of action. In order to investigate the cerebral distribution and kinetics of THA in the human brain in vivo, we performed positron emission tomography (PET) imaging with [11C]N-methyl-tetrahydro-aminoacridine (MTHA) in healthy human volunteers. After intravenous injection, [11C]MTHA crossed the blood-brain barrier and reached its maximum uptake between 10 and 40 minutes, depending on the brain regions. Uptake was higher in the grey matter structures, and lower in the white matter. After this peak, the radioactivity remained quasi- constant until 60 minutes in all regions with a half-life varying from 2.44 hours in the thalamus to 3.42 hours in the cerebral cortex. The ratios of regional to whole cerebral cortex brain radioactivity calculated between 50 and 70 minutes after the tracer injection were 1.14 +/- 0.04, 1.07 +/- 0. 03 and 1.06 +/- 0.04 in the putamen, cerebellum and thalamus, respectively. Overall, these results show that: (1) [11C]MTHA crosses the blood-brain barrier easily and is highly concentrated in the brain; (2) the regional brain distribution of [11C]MTHA does not parallel that of in vivo acetylcholinesterase (AChE) concentrations; and (3) the cerebral kinetics of [11C]MTHA are consistent with known plasmatic pharmacokinetics of THA in AD patients. We conclude that PET imaging with [11C]MTHA is a useful method for assessing the cerebral distribution and kinetics of THA in vivo.
We report here the first positron emission tomography (PET) images showing the in vivo regional distribution of acetylcholinesterase (AChE) in human brain. The study was carried out in eight healthy human volunteers using as a tracer [11C]-physostigmine ([11C]PHY), an inhibitor of AChE. After intravenous injection of [11C]PHY, radioactivity was rapidly taken up in brain tissue and reached maximal uptake within a few minutes, following a regional pattern mostly related to cerebral perfusion. After the peak, the cerebral radioactivity gradually decreased with a half-life varying from 20 to 35 min, depending on the brain structure. [11C] PHY retention was higher in regions rich in AChE, such as the striatum (half-life, 35 min), than in regions poor in AChE, such as the cerebral cortex (half-life, 20 min). At later times (25-35 min postinjection), the cerebral distribution of [11C]PHY was typical of AChE activity: putamen-caudate > cerebellum > brainstem > thalamus > cerebral cortex, with a striatal to cortex ratio of 2. These results suggest that PET studies with [11C]PHY can provide in vivo brain mapping of human AChE and are promising for the study of changes in AChE levels associated with neurodegenerative diseases.
Changes in the axonal transport of acetylcholinesterase (AChE) were studied in the painful mononeuropathy induced by setting 4 loose ligatures around the right sciatic nerve of the rat. Since changes in the axonal transport of AChE can be used to assess axonal degeneration/regeneration, we used this marker to investigate whether the time course of pain-related behavioral disorders observed following chronic constriction injury (CCI) to the sciatic nerve are related to the time course of the regeneration of the injured axons. In addition, a comparison was made between changes in AChE observed in this model of nerve injury and those observed after sciatic nerve crush. The rats were examined for pain-related disorders daily during the first postoperative week then at 7, 14 and 21 days after nerve ligation. The pain-related disorders, only detected from 7 days after ligation, were maximal at 14 days postinjury, and began to lessen at the end of the 3rd postoperative week. Within the first 3 days after loose ligation, the AChE transport dropped to 40% of its normal value, but recovered rapidly during the 3rd week post-surgery, indicating that most of the injured neurons were reconnecting their target cells. Thus, the injury produced by the loose ligatures was registered by the neurons several days before the first nociceptive manifestations of the injury, and the pain-related disorders lasted after most of the re-elongating axons had reconnected their target.
The cerebral distribution of [11C]physostigmine, an acetylcholinesterase inhibitor, was studied with autoradiography in rats and positron emission tomography in primates. In rat brain [11C]physostigmine radioactivity was exactly superimposable to acetylcholinesterase activity, being highest in the basal ganglia, moderate in the cortex and hippocampus, and low in the cerebellum. In primate brain, the early blood-flow dependent distribution of [11C]physostigmine was followed by a rapid redistribution to acetylcholinesterase-rich regions such as the striatum. The cerebral uptake of [11C]physostigmine was significantly reduced by competition with an excess of unlabeled physostigmine. These results suggest that [11C]physostigmine is a promising new ligand for in vivo imaging of acetylcholinesterase activity with PET.
THA (1,2,3,4-tetrahydro-9-amino-acridine, tacrine), a potential therapeutic agent for patients suffering from Alzheimer's disease, has multiple pharmacological sites of action in the brain. In order to study the cerebral binding sites of THA in vivo, we labeled a close derivative of THA with carbon 11 for positron emission tomography (PET) analysis. We report the biodistribution of this compound, 1,2,3,4-tetrahydro-9-[11C]methylaminoacridine ([11C]MTHA), in the rodent and describe the first PET experiments in non-human primates. The distribution of [11C]MTHA in baboon brain, although rather diffuse in the gray matter, showed a higher concentration in the cortex and basal ganglia than in the cerebellum and binding could be displaced (50%) by cold THA. These results suggest that [11C]MTHA is a promising PET ligand for the study of the cerebral binding of THA.
Aging in the sciatic nerve of the rat is characterized by various alterations, mainly cytoskeletal impairment, the presence of residual bodies and glycogen deposits, and axonal dystrophies. These alterations could form a mechanical blockade in the axoplasm and disturb the axoplasmic transports. However, morphometric studies on the fiber distribution indicate that the increase of the axoplasmic compartment during aging could obviate this mechanical blockade. Analysis of the axoplasmic transport, using acetylcholinesterase (AChE) molecular forms as markers, demonstrates a reduction in the total AChE flow rate, which is entirely accounted for by a significant bidirectional 40-60% decrease in the rapid axonal transport of the G4 molecular form. However, the slow axoplasmic flow of G1 + G2 forms, as well as the rapid transport of the A12 form of AChE, remain unchanged. Our results support the hypothesis that the alterations observed in aged nerves might be related either to the impairment in the rapid transport of specific factor(s) or to modified exchanges between rapidly transported and stationary material along the nerves, rather than to a general defect in the axonal transport mechanisms themselves.
Title: Axonal transport of the molecular forms of acetylcholinesterase in developing and regenerating peripheral nerve Couraud JY, Di Giamberardino L, Hassig R, Mira JC Ref: Experimental Neurology, 80:94, 1983 : PubMed
In chick sciatic nerve, acetylcholinesterase (AChE) occurs in four main molecular forms characterized by their sedimentation coefficients in sucrose gradients, referred to as G1 (5S), G2 (7.5S), G4 (11S), and A12 (20S). Under normal conditions, we previously showed by accumulation technique that the G4 and A12 forms are rapidly transported along the axons, whereas G1 and G2 are carried much more slowly. Here, we used to the same technique to study the anterograde axonal transport of these different AChE forms during normal axonal growth and experimental regeneration. During the first 2 months after hatching, G4 and A12 transport virtually doubled, whereas G1 + G2 transport increased only slightly. After nerve cutting, crushing, or freezing, the flow rates of G1 + G2 and G4 in the regenerating proximal stump decreased by 75% at 4 to 7 days compared with control values and that of A12, by 90 to 95%. In crushed and frozen nerves the transport of all four AChE forms slowly recovered thereafter, but failed to attain control values even after 7 weeks. In cut nerves, on the contrary, no significant recovery of G1 + G2, or G4 transport occurred, but A12 transport began to recover by day 7. Taken together, our results show that axonal transport of G1 + G2, G4, and A12 is selectively regulated in chick sciatic nerve, and suggest that the A12 form of AChE might have a special role and/or destination in regenerating axons.
During the development of diabetic neuropathy in the mouse C57BL/Ks (db/db), the axonal transport of AChE molecular forms was tested in the sciatic nerve, by measuring the accumulation of enzyme activity in front of a nerve transection. No alteration of the fast flow rate of G4 and A12 molecular forms was found until 220 days of age. On the other hand, a reduced flow rate of G1 and G2 molecular forms, probably conveyed by slow axoplasmic flow, was noticed in the late phase of diabetic neuropathy. This result is consistent with the view that axonal dwindling could be related to disturbances of slow axonal transport and that the reduction in conduction velocity, observed at an earlier stage, may be due to other causes.
Title: Slow axonal transport of the molecular forms of butyrylcholinesterase in a peripheral nerve Couraud JY, Di Giamberardino L, Hassig R Ref: Neuroscience, 7:1015, 1982 : PubMed
Butyrylcholinesterase was found in chick sciatic nerve in four main molecular forms--G1, G2, G4 and A12--distinguishable by thier sedimentation coefficients in sucrose gradients (4.2S, 6.4S, 11.3S and 19S, respectively). Axonal transport of butyrylcholinesterase was studied by measuring the accumulation of its molecular forms on each side of a transected sciatic nerve. Twenty-four hours after transection, butyrylcholinesterase activity had risen by about 32% at the extremity of the proximal stump, and by 20% at the extremity of the distal stump. Proximal accumulation was due to a two-fold rise in G4 activity and to a six-fold rise in A12 activity, whereas distal accumulation was exclusively due to a 50% increase in G4 activity, accompanied by the complete loss of A12. The activities of G1 and G2 remained stable in both directions. Under our experimental conditions, the accumulation of butyrylcholinesterase activity cannot be attributable to local protein synthesis, cross-contamination with accumulated acetylcholinesterase or the presence of plasma butyrylcholinesterase. Hence we conclude that all A12 butyrylcholinesterase molecules were carried in the anterograde direction, moving at 11.6 +/- 4.2 mm/day, and that probably some of the G4 molecules were slowly transported in both directions. These findings suggest that some of the butyrylcholinesterase is located in the axonal mitochondria and/or axolemma.
Acetylcholinesterase (AChE) is present in nervous and muscular tissues of normal chickens in four main molecular forms (G1, G2, G4, and A12), distinguishable by sedimentation analysis. In the sciatic nerve of acrylamide-poisoned chickens, the anterograde axonal transport of A12 AChE was reduced by 60%, and that of G4 by 21%, compared to control values whereas the slow axoplasmic transport of G1 and G2 was unaffected. Regarding the leg muscles, only the tibialis anterior revealed dramatic alterations in the distribution of it AChE forms coinciding with a large reduction in the number of nerve endings. In acrylamide poisoning, the AChE molecular forms were considered as very sensitive markers of both axonal transport phases and of the innervation state. Our results support the hypothesis that a defect in the fast axonal transport of proteins might be involved in the degeneration process of the disease.
The levels and molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and pseudocholinesterase (psiChE, EC 3.1.1.8) were examined in various skeletal muscles, cardiac muscles, and neural tissues from normal and dystrophic chickens. The relative amount of the heavy (Hc) form of AChE in mixed-fibre-type twitch muscles varies in proportion to the percentage of glycolytic fast-twitch fibres. Conversely, muscles with higher levels of oxidative fibres (i.e., slow-tonic oxidative-glycolytic fast-twitch, or oxidative slow-twitch) have higher proportions of the light (L) form of AChE. The effects of dystrophy on AChE and psiChE are more severe in muscles richer in glycolytic fast-twitch fibres (e.g., pectoral or posterior latissimus dorsi, PLD); there is no alteration of AChE or psiChE in a slow-tonic muscle. In the pectoral of PLD muscles from older dystrophic chickens, however, the AChE forms revert to a normal distribution while the pesChE pattern remains abnormal. Muscle psiChE is sensitive to collagenase in a similar way as is AChE, thus apparently having a similar tailed structure. Unlike skeletal muscle, cardiac muscle has very high levels of psiChE, present mainly as the L form; AChE is present mainly as the medium (M) form, with smaller amounts of L and Hc. The latter pattern of AChE forms resembles that seen in several neural tissues examined. No alterations in AChE or psiChE were found in cardiac or neural tissues from dystrophic chickens.
Title: Acetylcholinesterase and Butyrylcholinesterase: Similarities in Normal and Denervated Muscles, Differences in Axonal Transport Di Giamberardino L, Couraud JY Ref: Advances in Behavioral Biology, 25:387, 1981 : PubMed
Title: Contribution of axonal transport to the renewal of myelin phospholipids in peripheral nerves. I. Quantitative radioautographic study Droz B, Di Giamberardino L, Koenig HL Ref: Brain Research, 219:57, 1981 : PubMed
Kinetics of phospholipid constituents transferred from the axon to the myelin sheath were studied in the oculomotor nerve (OMN) and the ciliary ganglion (CG) of chicken. Axons of the OMN were loaded with transported phospholipids after an intracerebral injection of [2-3H]glycerol or [3H]labeled choline. Quantitative electron microscope radioautography revealed that labeled lipids were transported in the axons mainly associated with the smooth endoplasmic reticulum. Simultaneously, the labeling of the myelin sheath was found in the Schmidt-Lanterman clefts and the inner myelin layers. The outer Schwann cell cytoplasm and the outer myelin layers contained some label with [methyl-3H]choline, but virtually none with [2-3H]glycerol. With time the radioactive lipids were redistributed throughout and along the whole myelin sheath. Since [2-3H]glycerol incorporated into phospholipids is practically not re-utilized, the occurrence of label in myelin results from a translocation of entire phospholipid molecules and from their preferential insertion into Schmidt-Lanterman clefts. In this way, the axon-myelin transfer of phospholipid contributes rapidly to the renewal of a limited pool of phospholipids in the inner myelin layers. When [methyl-3H]choline was used as precursor of phospholipids, the rapid appearance of the label in the inner myelin layers was interpreted also as an axon-myelin transfer of labeled phospholipids. However, the additional labeling of the outer Schwann cell cytoplasm adjacent to Schmidt-Lanterman clefts and of the outer myelin layers reflects a local re-incorporation of the base released from the axon. By these two processes, the axon contributes to purvey the inner myelin layers with new phospholipids and the Schwann cells with new choline molecules.
Title: Acetylcholinesterase molecular forms in chick ciliary ganglion: pre- and postsynaptic distribution derived from denervation, axotomy, and double section Couraud JY, Koenig HL, Di Giamberardino L Ref: Journal of Neurochemistry, 34:1209, 1980 : PubMed
Title: Axonal transport of the molecular forms of acetylcholinesterase in chick sciatic nerve Couraud JY, Di Giamberardino L Ref: Journal of Neurochemistry, 35:1053, 1980 : PubMed
Acetylcholinesterase (AChE) polymorphism was studied in the sciatic nerve of 4-week-old Leghorn chicks, by sucrose gradient sedimentation analysis. Four main AChE molecular forms were found with sedimentation coefficients of 5S, 7.5S, 11.5S and 20S respectively. Axonal transport of each of these forms was investigated on the basis of the enzyme accumulation kinetics measured on both sides of nerve transections and of the enzyme redistribution kinetics in nerve segments isolated in vivo. After nerve transection, 11.5S and 20S forms accumulated faster in the anterograde than in the retrograde direction and also much faster than 5S and 7.5S forms in the anterograde direction. Retrograde accumulations of 5S and 7.5S were faint or negligible. In addition, 1 h after nerve cutting, the accumulation rates for 11.5S and 20S forms (but not for 5S and 7.5S) fell, in both directions, to about one-third of their initial values, probably owing to reversal of axonal transport at the axotomy site. Local protein synthesis inhibition by cycloheximide did not affect the accumulation of 11.5S and 20S in front of a transection, at least during the first hours, but reduced that of 5S and 7.5S by about 40%. In isolated nerve segments in vivo, the rapidly mobile fraction of AChE was estimated to constitute 23% of the total enzyme activity present in the nerve, 14% of it moving in an anterograde and 9% in a retrograde direction. A small amount of 11.5S molecules (approx. 20%) was in rapid transit (two-thirds in the anterograde and one-third in the retrograde direction), whereas almost all the 20S--about 90%--migrated rapidly (two-thirds forwards and one-third backwards). Anterograde velocities of 408 +/- 94 and 411 +/- 161 mm/day respectively were estimated for the 11.5S and 20S forms. Their respective retrograde velocities were 175 +/- 85 and 145 +/- 107 mm/day. Assuming that the totality of 5S and 7.5S molecules are moving in the anterograde direction, their accumulation rates were consistent with the average anterograde velocities of 2.9 +/- 1.3 and 5.1 +/- 1.4 mm/day, respectively.
Title: [Molecular forms of acetylcholinesterase in the chicken ciliary ganglion; changes after denervation, axotomy and double section] Couraud JY, Koenig H, Di Giamberardino L Ref: Comptes Rendus de l Academie des Sciences, 288:1199, 1979 : PubMed
Four main molecular forms of acetylcholinesterase (AChE), with sedimentation coefficients of 5, 7.5, 11.5 and 20 S, are found in Chicken ciliary ganglion. After denervation, the loss in 11.5 and 20 S forms occuring in 48 hrs coincides with the disappearance of presynaptic structures. In contrast, axotomy induces an early and durable increase in 7.5 S form. From these results, it is inferred that 11.5 and 20 S forms are predominant in presynaptic structures and 7.5 S form is mainly postsynaptic. In addition, the effects observed after simultaneous denervation and axotomy show a reciprocal control between pre- and postsynaptic elements. Finally, a trans-synaptic effect is exerted on 20 S AChE in controlateral ganglion after preganglionic sections.
Title: Normal axonal transport of acetylcholinesterase forms in peripheral nerves of dystrophic chickens Di Giamberardino L, Couraud JY, Barnard EA Ref: Brain Research, 160:196, 1979 : PubMed
Title: Axon-myelin transfer of phospholipid components in the course of their axonal transport as visualized by radioautography Droz B, Di Giamberardino L, Koenig HL, Boyenval J, Hassig R Ref: Brain Research, 155:347, 1978 : PubMed
Title: Axonal migration of protein and glycoprotein to nerve endings. II. Radioautographic analysis of the renewal of glycoproteins in nerve endings of chicken ciliary ganglion after intracerebral injection of (3H)fucose and (3H)-glucosamine Bennett G, Di Giamberardino L, Koenig HL, Droz B Ref: Brain Research, 60:129, 1973 : PubMed
Title: Axonal migration of protein and glycoprotein to nerve endings. 3. Cell fraction analysis of chicken ciliary ganglion after intracerebral injection of labeled precursors of proteins and glycoproteins Di Giamberardino L, Bennett G, Koenig HL, Droz B Ref: Brain Research, 60:147, 1973 : PubMed
Title: Axonal migration of protein and glycoprotein to nerve endings. I. Radioautographic analysis of the renewal of protein in nerve endings of chicken ciliary ganglion after intracerebral injection of (3H)lysine Droz B, Koenig HL, Biamberardino LD, Di Giamberardino L Ref: Brain Research, 60:93, 1973 : PubMed
