Reorganization of axoplasmic organelles following beta, beta'- iminodipropionitrile administration

beta, beta'-Iminodipropionitrile (IDPN), a synthetic compound that selectively impairs slow axonal transport, produced a rearrangement of the axonal cytoskeleton, smooth endoplasmic reticulum, and mitochondria. Immunoperoxidase staining using an antiserum to the 68,000-dalton neurofilament subunit demonstrated a displacement of neurofilaments toward the periphery of the axons of IDPN-treated rats. This change occurred simultaneously along the entire length of the sciatic nerve. Ultrastructural morphometry of the axonal organelles confirmed the peripheral relocation of neurofilaments and also showed a displacement of microtubules, smooth endoplasmic reticulum, and mitochondria to the center of the axons. The overall density of axonal mitochondria was increased, whereas those of other organelles were not significantly changed. Axons were reduced in size by 10--24%, the large axons being more affected than the small ones. The observed rearrangement of axonal organelles may be due to an effect of IDPN on microtubule-neurofilament interactions, which could in turn explain the impairment of the slow transport. Axons in IDPN intoxication are a useful model to study the organization of the axoplasm and the mechanism of axonal transport.     

β,β''-Iminodipropionitrile Impairs Retrograde Axonal Transport

beta,beta'-Iminodipropionitrile (IDPN), a neurotoxin, causes redistribution of neurofilaments in axons followed by the development of proximal axonal swellings and, in chronic intoxication, a distal decrease in axonal caliber. The latter changes are caused by a selective impairment in the slow anterograde axonal transport of neurofilament proteins. To assess the role of retrograde axonal transport in IDPN toxicity, we used [3H]N-succinimidyl propionate ([3H]NSP) to label covalently endogenous axonal proteins in sciatic nerve of the rat and measured the accumulation of radioactively labeled proteins in the cell bodies of motor and sensory neurons over time. IDPN was injected intraneurally 6 h or intraperitoneally 1 day before subepineurial injection of [3H]NSP into the sciatic nerve, and the animals were killed 1, 2, and 7 days after [3H]NSP injection. Neurotoxicity was assessed by electron microscopic observation of the nerves of similarly treated animals. Both intraneural and intraperitoneal injection of IDPN caused an acute reduction in the amount of labeled proteins transported back to the cell bodies. The early appearance of these changes suggests that alterations in retrograde transport may play a role in the production of the neuropathic changes.     

A Stochastic Multiscale Model That Explains the Segregation of Axonal Microtubules and Neurofilaments in Neurological Diseases

The organization of the axonal cytoskeleton is a key determinant of the normal function of an axon, which is a long thin projection of a neuron. Under normal conditions two axonal cytoskeletal polymers, microtubules and neurofilaments, align longitudinally in axons and are interspersed in axonal cross-sections. However, in many neurotoxic and neurodegenerative disorders, microtubules and neurofilaments segregate apart from each other, with microtubules and membranous organelles clustered centrally and neurofilaments displaced to the periphery. This striking segregation precedes the abnormal and excessive neurofilament accumulation in these diseases, which in turn leads to focal axonal swellings. While neurofilament accumulation suggests an impairment of neurofilament transport along axons, the underlying mechanism of their segregation from microtubules remains poorly understood for over 30 years. To address this question, we developed a stochastic multiscale model for the cross-sectional distribution of microtubules and neurofilaments in axons. The model describes microtubules, neurofilaments and organelles as interacting particles in a 2D cross-section, and is built upon molecular processes that occur on a time scale of seconds or shorter. It incorporates the longitudinal transport of neurofilaments and organelles through this domain by allowing stochastic arrival and departure of these cargoes, and integrates the dynamic interactions of these cargoes with microtubules mediated by molecular motors. Simulations of the model demonstrate that organelles can pull nearby microtubules together, and in the absence of neurofilament transport, this mechanism gradually segregates microtubules from neurofilaments on a time scale of hours, similar to that observed in toxic neuropathies. This suggests that the microtubule-neurofilament segregation can be a consequence of the selective impairment of neurofilament transport. The model generates the experimentally testable prediction that the rate and extent of segregation will be dependent on the sizes of the moving organelles as well as the density of their traffic.     

Differential turnover of phosphate groups on neurofilament subunits in mammalian neurons in vivo

The phosphorylation and dephosphorylation of specific proteins was demonstrated directly in the intact vertebrate nervous system in vivo. By exploiting the neurons' ability to segregate a select group of cytoskeletal proteins from most other phosphorylated constituents of the cell by axoplasmic transport, we were able to examine the dynamics of phosphate turnover on neurofilament proteins in mouse retinal ganglion cell neurons simultaneously labeled with [32P]orthophosphate and [3H]proline in vivo. Three [3H]proline-labeled neurofilament protein (NFP) subunits, designated H (160-200 kDa), M (135-145 kDa), and L (68-70 kDa), entered optic axons in a mole:mole ratio similar to that of isolated axonal neurofilaments, supporting the notion that newly synthesized NFPs are transported along axons as assembled neurofilaments. NFP subunits incorporated high levels of 32P before reaching axonal sites at the level of the optic nerve. As neurofilaments were transported along axons, however, many initially incorporated [32P]phosphate groups were removed. Loss of these phosphate groups occurred to a different extent on each subunit. A minimum of 50-60 and 35-40% of the labeled phosphate groups was removed in a 5-day period from the L and M subunits, respectively. By contrast, the H subunit exhibited relatively little or no phosphate turnover during the same period. Dephosphorylation of L in axons is accompanied by a decrease in its net state of phosphorylation; changes in the phosphorylation state of H and M, however, also reflect ongoing addition of phosphates to these polypeptides during axonal transport (Nixon, R.A., Lewis, S.E., and Marotta, C.A. (1986) J. Neurosci., in press). The possibility is raised that dynamic rearrangements of phosphate topography on NFPs represent a mechanism to coordinate interactions of neurofilaments with other proteins as these elements are transported and incorporated into the stationary cytoskeleton along retinal ganglion cell axons.     

Overexpression of Tau Protein Inhibits Kinesin-dependent Trafficking of Vesicles,Mitochondria, and Endoplasmic Reticulum: Implications for Alzheimer's Disease

The neuronal microtubule-associated protein tau plays an important role in establishing cell polarity by stabilizing axonal microtubules that serve as tracks for motor-protein–driven transport processes. To investigate the role of tau in intracellular transport, we studied the effects of tau expression in stably transfected CHO cells and differentiated neuroblastoma N2a cells. Tau causes a change in cell shape, retards cell growth, and dramatically alters the distribution of various organelles, known to be transported via microtubule-dependent motor proteins. Mitochondria fail to be transported to peripheral cell compartments and cluster in the vicinity of the microtubule-organizing center. The endoplasmic reticulum becomes less dense and no longer extends to the cell periphery. In differentiated N2a cells, the overexpression of tau leads to the disappearance of mitochondria from the neurites. These effects are caused by tau''s binding to microtubules and slowing down intracellular transport by preferential impairment of plus-end–directed transport mediated by kinesin-like motor proteins. Since in Alzheimer''s disease tau protein is elevated and mislocalized, these observations point to a possible cause for the gradual degeneration of neurons.     

Early Effects of β,β'-Iminodipropionitrile on Tubulin Solubility and Neurofilament Phosphorylation in the Axon

Abstract: To elucidate the role of neurofilaments in microtubule stabilization in the axon, we studied the effects of β,β'-iminodipropionitrile (IDPN) on the solubility and transport of tubulin as well as neurofilament phosphorylation in the motor fibers of the rat sciatic nerve. IDPN is known to impair the axonal transport of neurofilaments, causing accumulation of neurofilaments in the proximal axon and segregation of neurofilaments to the peripheral axoplasm throughout the nerve. Administration of IDPN at various intervals after radioactive labeling of the spinal cord with l -[³⁵S]methionine revealed that transport inhibition occurred all along the nerve within 1–2 days. Transport of cold-insoluble tubulin, which accounts for 50% of axonal tubulin, was also affected. A significant increase in the proportion of cold-soluble tubulin was observed, reaching a maximum at 3 days after IDPN treatment and returning to the control level in the following weeks. Preceding this change in tubulin solubility, a transient decrease in the phosphorylation level of the 200-kDa neurofilament protein was detected in the ventral root using phosphorylation-dependent antibodies. These early changes agreed in timing with the onset of segregation and transport inhibition, suggesting that interaction between neurofilaments and microtubules possibly regulated by phosphorylation plays a significant role in microtubule stabilization.     

Cytoskeletal architecture and immunocytochemical localization of microtubule-associated proteins in regions of axons associated with rapid axonal transport: the beta,beta''-iminodipropionitrile-intoxicated axon as a model system

Axons from rats treated with the neurotoxic agent beta,beta'-iminodipropionitrile (IDPN) were examined by quick-freeze, deep-etch electron microscopy. Microtubules formed bundles in the central region of the axons, whereas neurofilaments were segregated to the periphery. Most membrane-bounded organelles, presumably including those involved in rapid axonal transport, were associated with the microtubule domain. The high resolution provided by quick-freeze, deep-etch electron microscopy revealed that the microtubules were coated with an extensive network of fine strands that served both to cross-link the microtubules and to interconnect them with the membrane-bounded organelles. The strands were decorated with granular materials and were irregular in dimension. They appeared either singly or as an extensive anastomosing network in fresh axons. The microtubule-associated strands were observed in fresh, saponin-extracted, or aldehyde-fixed tissue. To explore further the identity of the microtubule-associated strands, microtubules purified from brain tissue and containing the high molecular weight microtubule-associated proteins MAP 1 and MAP 2 were examined by quick-freeze, deep-etch electron microscopy. The purified microtubules were connected by a network of strands quite similar in appearance to those observed in the IDPN axons. Control microtubule preparations consisting only of tubulin and lacking the MAPs were devoid of associated strands. To learn which of the MAPs were present in the microtubule bundles in the axon, sections of axons from IDPN-treated rats were examined by immunofluorescence microscopy using antibodies to MAP 1A, MAP 1B, MAP 2, and tubulin. Anti-MAP 2 staining was only marginally detectable in the IDPN-treated axons, consistent with earlier observations. Anti-MAP 1A and anti-MAP 1B brightly stained the IDPN-treated axons, with the staining exclusively limited to the microtubule domains. Furthermore, thin section-immunoelectron microscopy using colloidal gold-labeled second antibodies revealed that both anti-MAP 1A and anti-MAP 1B stained fuzzy filamentous structures between microtubules. In view of earlier work indicating that rapid transport is associated with the microtubule domain in the IDPN-treated axon, it now appears that MAP 1A and MAP 1B may play a role in this process. We believe that MAP 1A and MAP 1B are major components of the microtubule-associated fibrillar matrix in the axon.     

The role of calcium in the initiation of fast axonal transport

Incubation of neuronal cell bodies in a calcium-free medium depresses the amount, but not the rate, of fast axonal transport of [3H]protein. Under these conditions, which do not affect protein synthesis or general energy metabolism, less protein appears to be loaded onto the transport system. Depression of transport also is seen when cell bodies are exposed to medium containing Co2+; selective exposure of axons to this medium has no effect on transport. These findings have led to the concept of an initiation phase of fast axonal transport that comprises the events by which selected proteins are transferred from their polysomal sites of synthesis to the transport system. The divalent cation specificity of the Ca2+ requirement, and its occurrence subsequent to Golgi apparatus-associated glycosylation, suggest that proteins destined for fast axonal transport are routed through the soma in a manner similar to that of secretory proteins and integral membrane proteins in nonneural cells. This analogy is pursued to consider a scheme whereby Golgi-derived vesicles deliver fast-transported proteins to the axonal smooth endoplasmic reticulum. Possible roles of Ca2+ in the formation and exocytotic fusion of such vesicles are considered.     

ULTRASTRUCTURE AND FUNCTION OF GROWTH CONES AND AXONS OF CULTURED NERVE CELLS

Dorsal root ganglion nerve cells undergoing axon elongation in vitro have been analyzed ultrastructurally. The growth cone at the axonal tip contains smooth endoplasmic reticulum, vesicles, neurofilaments, occasional microtubules, and a network of 50-A in diameter microfilaments. The filamentous network fills the periphery of the growth cone and is the only structure found in microspikes. Elements of the network are oriented parallel to the axis of microspikes, but exhibit little orientation in the growth cone. Cytochalasin B causes rounding up of growth cones, retraction of microspikes, and cessation of axon elongation. The latter biological effect correlates with an ultrastructural alteration in the filamentous network of growth cones and microspikes. No other organelle appears to be affected by the drug. Removal of cytochalasin allows reinitiation of growth cone-microspike activity, and elongation begins anew. Such recovery will occur in the presence of the protein synthesis inhibitor cycloheximide, and in the absence of exogenous nerve growth factor. The neurofilaments and microtubules of axons are regularly spaced. Fine filaments indistinguishable from those in the growth cone interconnect neurofilaments, vesicles, microtubules, and plasma membrane. This filamentous network could provide the structural basis for the initiation of lateral microspikes and perhaps of collateral axons, besides playing a role in axonal transport.     

Fast axonal transport in motor nerve regeneration

This report describes the fast axonal transport of [³H]-leucine-labeled proteins in regenerating rat sciatic motor nerves. A normal rate of fast transport (383 ± 33 mm/day) was present in the regenerating sprouts, as well as in the central stumps. The rapidly transported proteins passed the level of axotomy without impediment, and accumulated in the endings of the regenerating sprouts, as shown by electron microscope autoradiography. In addition, transported proteins accumulated in terminal neuromas. The relative amount of protein-incorporated radioactivity in the crest of fast transport in the regenerating nerves was increased compared to control nerves. These results are interpreted to suggest that the mechanism of fast transport is the same in regenerating sprouts as in normal axons; during regeneration fast transport appears to add newly synthesized materials to the growing tip.     

Early structural changes in the axoplasmic cytoskeleton after axotomy studied by cryofixation

Summary Alterations in the cytoskeleton were studied in the axoplasm of neurites at the tips of proximal stumps of transected chicken sciatic nerves. The studies were carried out using cryofixation with a nitrogen-cooled propane jet. The most immediate effect is the almost complete disassembly of axoplasmic microtubules. This consequently causes the axonal transport of membrane-bounded organelles to cease and results in an accumulation of mitochondria and vesicles of the smooth endoplasmic reticulum. The neurofilament network is partially disorganized. Neurofilaments become shorter and fragmented, and are linked by a large number of anastomosed cross-linkers. The neurofilaments become newly aligned to the axis of the axoplasm and are of normal length 48–72 h after the transsection. At this stage the newly formed neurofilament bundles are in close proximity to the anastomosed cisternae and profiles of the smooth endoplasmic reticulum. The axonal sprouts always show a normally organized cytoskeletal network. These studies support the idea that the rapid remodelling of the neurofilament network is apparently a local event, not dependent on the slow transport of cytoskeletal materials to the tip of the proximal stump. The repair of the degraded cytoskeleton may be in accordance with the function of the endoplasmic reticulum as Ca²⁺-sequestering membrane system, which may be involved in restoring the physiological conditions of the axoplasm.     

[32P]orthophosphate and [35S]methionine label separate pools of neurofilaments with markedly different axonal transport kinetics in mouse retinal ganglion cells in vivo

Newly synthesized neurofilament proteins become highly phosphorylated within axons. Within 2 days after intravitreously injecting normal adult mice with [³²P]orthophosphate, we observed that neurofilaments along the entire length of optic axons were radiolabeled by a soluble³²P-carrier that was axonally transported faster than neurofilaments.³²P-incorporation into neurofilament proteins synthesized at the time of injection was comparatively low and minimally influenced the labeling pattern along axons.³²P-incorporation into axonal neurofilaments was considerably higher in the middle region of the optic axons. This characteristic non-uniform distribution of radiolabel remained nearly unchanged for at least 22 days. During this interval, less than 10% of the total³²P-labeled neurofilaments redistributed from the optic nerve to the optic tract. By contrast, newly synthesized neurofilaments were selectively pulse-labeled in ganglion cell bodies by intravitreous injection of [³⁵S]methionine and about 60% of this pool translocated by slow axoplasmic transport to the optic tract during the same time interval. These findings indicate that the steady-state or resident pool of neurofilaments in axons is not identical to the newly synthesized neurofilament pool, the major portion of which moves at the slowest rate of axoplasmic transport. Taken together with earlier studies, these results support the idea that, depending in part on their phosphorylation state, transported neurofilaments can interact for short or very long periods with a stationary but dynamic neurofilament lattice in axons.Special issue dedicated to Dr. Sidney Ochs.     

Effects of lidocaine on axonal morphology,microtubules, and rapid transport in rabbit vagus In vitro

Treatment with lidocaine, at exposures which completely block impulse conduction and rapid axonal transport, resulted in a sequence of morphological effects on rabbit vagus never in vitro. Low exposures (0.3% for 90 min, 0.4% for 45 min, 0.6% for 25 min) caused a reorientation and proliferation of smooth endoplasmic reticulum (SER); the number of axonal microtubules either remained normal or was increased. Intermediate exposures (0.4% for 90 min, 0.6% for 45 min) Produced a similar effect on the SER, as well as causing a 50% reduction in microtubule number and some swelling of axons and Schwann cells. The highest exposure (0.6% for 75 min) caused over 90% reduction in microtubule number, a partial loss of neurofilaments, and severe swelling of axons, Schwann call, and mitochondria. Reversibility of these effects was tested in lidocaine-treated nerves that had been washed with fresh culture medium. There was good recovery of nerve exposures. After intermediate exposures, there was only partial return of rapid transport, even though imposures, there was morphological, and no recovery occured throughout the excised vagus nerve in efferent and afferent axons as well as Schwann cells, and these events did not coincide with changes in the functional state of rapid axonal transport.     

Observation of the proximal portions of axons of anterior-horn cells in the human spinal cord

The proximal portions of axons of large anterior-horn cells were investigated in the lumbar cords of 10 normal human autopsy cases. Light-microscopically, 81 myelinated axons were observed to be connected with the cell body. Of the 81 axons, 78 emanated from the cell body and 3 others originated in the proximal part of primary dendrites. As for normal-looking neurons (n = 77), the length of the axon hillock plus initial segment was 64.0 +/- 12.3 microns (average +/- SEM), ranging from 47.5 to 110.0 microns, while the diameter of the thinnest portion of the initial segment was 2.40 +/- 0.30 microns (average +/- SEM), ranging from 1.32 to 3.92 microns. Electron-microscopically, the predominant organelles of the axon hillock were mitochondria, neurofilaments which merged into the axon and occasional granular endoplasmic reticulum. A few synaptic boutons were found on the surface of the axon hillock. The cell membrane of the initial segment consisted of a layer of electron-dense material (undercoating). The cytoplasm contained many neurofilaments, running parallel to the longitudinal axis of the initial segment. Among the neurofilaments, lysosomes, smooth endoplasmic reticulum, dense bodies and vesicular profiles as well as mitochondria were seen. At the beginning of the myelin sheath, the axoplasm contained mitochondria, many neurofilaments and occasional lysosomes.     

DISTRIBUTION OF LEUCINE-3H DURING AXOPLASMIC TRANSPORT WITHIN REGENERATING NEURONS AS DETERMINED BY ELECTRON-MICROSCOPE RADIOAUTOGRAPHY

The distribution of leucine-³H in neurons was determined by electron-microscope radioautography after infusion of label into the spinal cord or sensory ganglia of regenerating newts. In the nerve cell bodies 3 days after infusion, the highest concentration of label per unit area occurred over the rough-surfaced endoplasmic reticulum. In the large brachial nerves, the silver grains were not distributed uniformly in the axoplasm, indicating that the labeled materials are restricted in their movement to certain regions of the axon. Almost all of the radioautographic grains observed in myelinated nerves could be accounted for by the presence of a uniformly labeled band occupying the area 1500–9000 A inside the axolemma. This region of the axon was rich in microtubules and organelles while the unlabeled central core of the axon contained mainly neurofilaments. This observation supports the hypothesis that microtubules are related to axonal transport. In small, vesicle-filled nerve terminals in the blastema, labeled material was restricted to a thin zone a short distance beneath the plasma membrane while the central region of the terminal was largely unlabeled. The peripheral pattern of labeling in the nerve endings is consistent with successive addition of newly synthesized proteins at the periphery of the growth cone and release of substances such as trophic factors at the nerve terminal.     

Importance of monovalent ions for the fast axonal transport of proteins

Proteins labeled with [35S]methionine or [3H]leucine were generated in vitro in bullfrog dorsal root ganglia and their fast axonal transport in the spinal nerves was followed during a subsequent incubation period. Incubation of the ganglia in a medium where sucrose, choline chloride, or sodium isethionate replaced NaCl caused respectively an 88, a 37, or a 76% reduction in the quantity of proteins carried by the fast axonal transport system; no decrease in synthesis of labeled proteins was observed and protein transport followed the usual time course. Incubation of desheathed spinal nerves in a medium where sucrose replaced NaCl reduced by 67% the quantity of labeled proteins which were transported past the desheathed region. Although both the axons and the dorsal root ganglia exhibit the requirement for monovalent ions to maintain fast axonal transport, the possibility that the ionic requirements of the ganglia pertain to the somal portion of the nerve cell is discussed.     

The long-term effects of a single injection of beta,beta'-iminodipropionitrile on slow axonal transport in the rat

Following a single intraperitoneal injection of beta,beta'-iminodipropionitrile (IDPN) simultaneously with or 5 weeks after injection of [35S]methionine into the ventral horn area of rat spinal cord, the changes of slowly migrating axonal proteins were analyzed electrophoretically up to 10 weeks after labeling, and the following results were obtained. After a single injection of IDPN, only the transport of neurofilament proteins is inhibited, leaving that of tubulin and actin almost unaffected, though a small portion of the former was retarded through the interaction with neurofilaments. The inhibitory effect of IDPN on neurofilament transport is not a complete blockage, but a slowing of the rate of transport to about a half of the control with a possible short halting period just after IDPN treatment. The dose-response data indicate a threshold between 0.5 and 1.0 g/kg of body weight, increasing the dose above which does not further affect the neurofilament transport. The transport of neurofilaments is uniformly impaired by IDPN along the whole axon.     

Cytoplasmic structure in rapid-frozen axons

Turtle optic nerves were rapid-frozen from the living state, fractured, etched, and rotary shadowed. Stereo views of fractured axons show that axoplasm consists of three types of longitudinally oriented domains. One type consists of neurofilament bundles in which individual filaments are interconnected by a cross-bridging network. Contiguous to neurofilament domains are domains containing microtubules suspended in a loose, granular matrix. A third domain is confined to a zone, 80-100 nm wide, next to the axonal membrane and consists of a dense filamentous network connecting the longitudinal elements of the axonal cytoskeleton to particles on the inner surface of the axolemma. Three classes of membrane-limited organelles are distinguished: axoplasmic reticulum, mitochondria, and discrete vesicular organelles. The vesicular organelles must include lysosomes, multivesicular bodies, and vesicles which are retrogradely transported in axons, though some vesicular organelles may be components of the axoplasmic reticulum. Organelles in each class have a characteristic relationship to the axonal cytoskeleton. The axoplasmic reticulum enters all three domains of axoplasm, but mitochondria and vesicular organelles are excluded from the neurofilament bundles, a distribution confirmed in thin sections of cryoembedded axons. Vesicular organelles differ from mitochondria in at least three ways with respect to their relationships to adjacent axoplasm: (a) one, or sometimes both, of their ends are associated with a gap in the surrounding granular axoplasm; (b) an appendage is typically associated with one of their ends; and (c) they are not attached or closely apposed to microtubules. Mitochondria, on the other hand, are only rarely associated with gaps in the axoplasm, do not have an appendage, and are virtually always attached to one or more microtubules by an irregular array of side-arms. We propose that the longitudinally oriented microtubule domains are channels within which organelles are transported. We also propose that the granular material in these channels may constitute the myriad enzymes and other nonfibrous components that slowly move down the axon.     

The effect of beta,beta'-iminodipropionitrile (IDPN) on cytoskeletal organization in cultured human skin fibroblasts

By impairing the axonal transport of neurofilaments, IDPN induces large, filament-filled swellings in the proximal portion of axons of spinal motor neurones. We have reported that 2,5-hexanedione, another agent producing focal accumulation of neurofilaments, induces aggregation of intermediate filaments of the vimentin type in human skin fibroblasts grown in tissue culture. IDPN was tested in this model to determine if this chemical also affects a general mechanism involved in cytoplasmic transport of the cytomatrix or a process exclusive to axonal flow of neurofilaments. Although aggregates of intermediate filaments were seen in fibroblasts exposed to IDPN, they did not occur in the absence of generalized cytotoxicity and were labelled by antisera against tubulin, actin and fibronectin in addition to vimentin. It is probable the effect of IDPN on the distribution of intermediate filaments was secondary to changes in cell shape occurring during slow detachment from the substratum.     

Inhibition of fast axonal transport in bullfrog nerves by dibenzazepine and dibenzocycloheptadiene calmodulin inhibitors

The effects of the calmodulin inhibitors amitriptyline, desipramine, imipramine, and clomipramine on fast axonal transport, oxidative metabolism, and density of axonal microtubules were measured in bullfrog spinal nerves in vitro. The four drugs tested inhibited the fast orthograde transport of [3H]leucine-labelled proteins and the fast retrograde transport of acetylcholinesterase at a concentration of 0.2 mM. Amitriptyline, desipramine, and imipramine were equipotent inhibitors of transport, and clomipramine was a more potent inhibitor than imipramine. The adenosine triphosphate content of the nerves was reduced by at most 19% by the compounds under study; such a reduction cannot account for the inhibition of fast axonal transport. Desipramine and imipramine had no significant effect on the density of microtubules in unmyelinated axons, whereas amitriptyline only reduced it by 18%; the inhibition of axonal transport by these three drugs can therefore not be explained by microtubule disruption. Clomipramine reduced microtubular density by 40%, and this effect may have contributed to the inhibition of fast axonal transport. The inhibition of fast axonal transport by desipramine, imipramine, and amitriptyline may be related to the inhibition of calmodulin function by these drugs. The similar potency of these three drugs as inhibitors of fast axonal transport goes in parallel with their known similar potency as calmodulin antagonists.