Golgi apparatus of the motor neurons in patients with amyotrophic lateral sclerosis and in mice models of amyotrophic lateral sclerosis

We examined the Golgi apparatus (GA) of motor neurons of patients with ALS and in mice models of ALS by immunohistological method using antiserum against MG160 and against components of the trans‐Golgi network (TGN46). The GA of half of the remaining spinal cord motor neurons of patients with sporadic ALS showed fragmentation, where the GA were dispersed or fragmented into numerous small, isolated elements. The GA of Betz cells in sporadic ALS were fragmented similar to that of anterior horn cells, and the GA of spinal cord motor neurons of those with familial ALS and of those with ALS with basophilic inclusions were fragmented or diminished. The GA in the majority of the motor neurons contained Bunina bodies, basophilic inclusions and superoxide dismutase 1 (SOD1)‐positive aggregates were fragmented. The motor neurons in transgenic mice expressing G93A mutation of the SOD1 gene showed the fragmentation of the GA months before the onset of paralysis. These findings suggest that the fragmentation of GA may be related to the neuronal degeneration in patients with ALS.     

Structural correlates of physiological abnormalities in β,β′-Iminodipropionitrile

β,β′-Iminodipropionitrile (IDPN) produces neurofilamentous giant axonal swellings in proximal internodes of large myelinated axons. Secondary demyelinative changes result from the production of these axonal enlargments. Electrophysiological studies have demonstrated profound alterations in the electrical properties of motor neurons (MN) within the spinal cord. On the basis of intracellular recordings, it has been suggested that electrical contacts may exist between swollen axons and neighboring MN. In addition, the possibility remained that synaptic contacts develop on demyelinated axonal swellings. In the present study, we report the lack of either synapses on demyelinated axonal swellings or direct electrical contacts between neighboring MN. Axonal swellings are surrounded by attenuated processes of glial cells (probably fibrillary astrocytes), a finding discussed in terms of its possible role in the production of ephaptic transmission. There was considerable variation in the degree of axonal enlargements and in the extent of secondary (passive and active) demyelination. It is suggested that these morphological changes may represent structural correlates of some electrophysiological alterations observed in IDPN neuropathy.     

Anterior horn cells with abnormal TDP-43 immunoreactivities show fragmentation of the Golgi apparatus in ALS

Recently, TAR DNA-binding protein of 43-kDa (TDP-43) was identified as a major component of ubiquitinated neuronal cytoplasmic inclusions observed in lower motor neurons in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions. We herein investigated the relationship between TDP-43 immunoreactivities and fragmentation of the Golgi apparatus (GA). Each mirror section of spinal cord tissues in 10 ALS and 3 control cases were immunostained with polyclonal anti-TDP-43 and polyclonal anti-trans-Golgi-network (TGN)-46 antibodies. The neurons were divided into subtypes according to differences in TDP-43 immunoreactivities, and we examined the morphological changes of GA in each type. We divided the neurons into four subtypes according to the observed differences in TDP-43 immunoreactivities, Type A: neurons showing normal nuclear staining, Type B: neurons showing a loss of normal nuclear staining and a few granular cytoplasmic immunoreactivities, Type C: neurons showing a lot of granular immunoreactivities and no inclusions, Type D: neurons with inclusions. All of the neurons in Type A showed normal GA profiles, however, almost all of the neurons with abnormal TDP-43 immunoreactivities (Type B–D) showed GA fragmentation. These results suggest that neurons with abnormal TDP-43 immunoreactivities are associated with dysfunction of the secretory pathway in motor neurons.     

Reduced expression of BTBD10 in anterior horn cells with Golgi fragmentation and pTDP‐43‐positive inclusions in patients with sporadic amyotrophic lateral sclerosis

Overexpression of BTBD10 (BTB/POZ domain‐containing protein 10) suppresses G93A‐superoxide dismutase 1 (SOD1)‐induced motor neuron death in a cell‐based amyotrophic lateral sclerosis (ALS) model. In the present study, paraffin sections of spinal cords from 13 patients with sporadic ALS and 10 with non‐ALS disorders were immunostained using a polyclonal anti‐BTBD10 antibody. Reduced BTBD10 expression in the anterior horn cells was more frequent in spinal cords from ALS patients than in cords from patients with non‐ALS disorders. We further investigated the relationship between the level of BTBD10 immunoreactivity and the morphology of the Golgi apparatus (GA) and the presence of phosphorylated TAR‐DNA‐binding protein 43 (pTDP‐43). Mirror sections of spinal cords from five sporadic ALS cases were immunostained with antibodies against BTBD10 and trans‐Golgi‐network (TGN)‐46 or pTDP‐43. Whereas 89.7–96.5% of the neurons with normal BTBD10 immunoreactivity showed normal GA morphology and no pTDP‐43 cytoplasmic aggregates, 86.2–94.3% of the neurons with reduced BTBD10 expression showed GA fragmentation and abnormal pTDP‐43 aggregates. These findings suggest that reduced BTBD10 expression is closely linked to the pathogenesis of sporadic ALS.     

Selective interruption of axonal transport of neurofilament proteins in the visual system by β,β′-iminodipropionitrile (IDPN) intoxication

β,β′-iminodipropionitrile (IDPN) is an agent that produces a marked impairment in the transport of neurofilaments. Its effect on other slowly transported cytoskeletal components sucas tubulin and actin is variable. Previous studies have evaluated transport of neurofilaments after IDPN intoxication in a neurofilament-ricsystem (sciatic motor nerves) and in a system devoid of neurofilaments (axons of the dorsal motor nucleus of the vagus). In the former, IDPN impairs the transport of tubulin and actin but to a lesser degree than it does neurofilament proteins. In the latter, tubulin and actin transport were not impaired, and neurofilament proteins were not present. In this study we evaluated the transport of the cytoskeletal components in a system witan intermediate amount of neurofilaments (the visual system). In the visual system, there is a selective and marked (50%) impairment in the transport of neurofilaments witno impairment in transport of tubulin or microtubule-associated proteins (tau group). We conclude that these different patterns of impairment in transport reflect the differences in pre-intoxication neurofilament content of the nerves examined, the effect of IDPN on the transport of the other components of slow transport being secondary to the presence of stagnated neurofilaments. This model also suggests that transport of neurofilaments can be selectively impaired without producing an effect on other major slow transport components.     

Immunohistochemical localization of 2′, 3′-cyclic nucleotide 3′-phosphodiesterase in the central nervous system

We describe the immunohistochemical localization for brain 2′, 3′-cyclic nucleotide 3′-phospho diesterase (EC 3.1.4.37) by the peroxidase-antiperoxidase method. Myelinated fibre tracts were immunohistochemically stained in 15-day-old rat cerebrum and spinal cord; at high magnification, myelinated nerve fibres and immature glial cells with their processes were shown to be stained.     

Binding of IDPN (β,β′-iminodipropionitrile) to rat spinal cord: Possible implication in the mechanism of spheroid formation in amyotrophic lateral sclerosis

β,β′-iminodipropionitrile (IDPN) produces ‘spheroids’ similar to those in certain cases of amyotrophic lateral sclerosis (ALS). Therefore, the target molecule of IDPN could be important to the understanding of the molecular mechanism of spheroid formation in ALS. Wistar rats were injected ip with ¹⁴C-labeled IDPN (¹⁴C-IDPN) and killed at 0.5, 1, 3, 6, 12 and 24 h thereafter. The radioactivity in each organ increased rapidly and reached the maximum at 0.5–1 h after ¹⁴C-IDPN injection. Thereafter, a rapid decrease occurred until 6 h, followed by a gradual decline until 24 h. The radioactivity in the cerebral cortex, diencephalon and cerebellum was higher than in the pons, medulla oblongata and spinal cord. Although high in the visceral organs and skeletal muscles, no or little radioactivity was detected in fat tissue. Autoradiography also confirmed these results. In three rats, ¹⁴C-IDPN was injected to the lumbar enlargement of the spinal cord. Six hours after injection, the segment was removed and homogenized with physiological saline (PS). After centrifugation, the supernatant was obtained (PS fraction). The pellet was resuspended with 4 mol/L urea and the supernatant was obtained (urea fraction). Each fraction was analysed by gel filtration. A peak of radioactivity was observed at the elution fraction Nos 19 and 20 (consistent with free ¹⁴C-IDPN) when PS fraction was applied. On application of urea fraction, another peak was obtained at the elution fractions Nos 8 and 9 (MW 60～80 kDa). The present study demonstrates that ¹⁴C-IDPN does not selectively accumulate to the spinal cord and suggests that an IDPN-binding molecule with an MW of 60–80 kDa is present in the spinal cord. The molecule may be related to the pathological process of spheroid formation in ALS.     

Novel axonal distribution of neurofilament‐H phosphorylated at the glycogen synthase kinase 3β‐phosphorylation site in its E‐segment

The Ser493 residue in the E‐segment of the rat neurofilament heavy chain (NF‐H) is phosphorylated by glycogen synthase kinase 3β (GSK3β) in vitro and in spinal cord. We examined Ser493 phosphorylation by analyzing developmental changes and cellular distribution of phospho‐Ser493 using phosphorylation‐site‐specific antibodies. This residue was phosphorylated in NF‐H prepared from human, rat, and mouse spinal cord, all species in which the amino acid sequence of NF‐H is known. Phosphorylated Ser493 appeared on postnatal day 2 in rat brain, at the same time when NF‐H is first detected. It gradually increased together with the increase in total NF‐H during brain development. Phospho‐Ser493 was detected on the phosphorylated form of NF‐H at multiple Lys‐Ser‐Pro (KSP) repeats, which are distributed mainly in axons. In rat ventral horn, phosphorylated Ser493 was localized in axons but not in cell bodies or dendrites. However, the distributions of phosphorylated Ser493 and KSP phosphorylation in axons were not identical. Ser493 was continuously phosphorylated at nodes of Ranvier, whereas the KSP sites were dephosphorylated. Ser493 was also phosphorylated in unmyelinated regions of optic nerve axons. A biochemical difference in phosphorylation between Ser493 and KSP repeats was also found; the subtle phosphorylation at Ser493 was detected in NF‐H unphosphorylated at the KSP repeats by immunoblotting cerebral cortex extracts. These results indicate that Ser493 in the NF‐H E‐segment is a novel site that is phosphorylated in both the myelinated and the unmyelinated regions of axons. © 2009 Wiley‐Liss, Inc.     

Localization of 2′,3′-cyclic nucleotide-3′-phosphohydrolase within the vertebrate retina

Histochemical and immunochemical techniques are used to locate 2',3'-cyclic nucleotide-3'-phosphohydrolase (2',3'-cNMP-3'ase) within cells of the vertebrate retina. A new histochemical method is described which links the hydrolysis of 2',3'-cNADP to the formation of a reduced, insoluble tetrazolium formazan. Photoreceptors from fish, bovine, and rat retinas are stained by this procedure. The reaction is blocked by 2'-AMP, a known inhibitor of 2',3'-cNMP-3'ase. Rabbit antibodies prepared against 2',3'-cNMP-3'ase from bovine brain are found to cross-react with bovine and rat retinal enzymes. Peroxidase-labeled antibody shows by light microscopy the greatest staining along the inner segments of the photoreceptors. Electron microscopy of the same preparations confirms binding to the plasma membrane of the inner segments of both rods and cones. Retinal 2',3'-cNMP-3'ase is thus predominantly associated with the photoreceptors, suggesting some role for 2',3'-cyclic nucleotides as substrates in visual function.     

Autoradiographic distribution of μ, δ and κ opioid binding sites in the superficial dorsal horn, over the rostrocaudal axis of the rat spinal cord

The purpose of this study was to use [3H]DAMGO, [3H]DTLET and [3H]EKC in the presence of 100 nM DAMGO and 100 nM DTLET, combined with a quantitative autoradiography to analyse the different proportions and the rostrocaudal distribution of mu, delta and kappa opioid binding sites in the superficial layers (laminae I and II) of the cervical (C6-C8), thoracic (T5-T7), lumbar (L3-L5) and sacral (S2-S3) dorsal horn of the rat. The proportions of the three main types of opioid binding sites, assessed by autoradiography in laminae I and II, were found homogeneous at each segmental level considered: 70.4-74.3%, 18.4-20.3% and 7.3-9.5% for mu, delta, kappa sites, respectively. The physiological relevance of these data is discussed.     

Coexpression of α2A‐adrenergic and δ‐opioid receptors in substance P‐containing terminals in rat dorsal horn

Agonists acting at α₂‐adrenergic and opioid receptors (α₂ARs and ORs, respectively) inhibit pain transmission in the spinal cord. When coadministered, agonists activating these receptors interact in a synergistic manner. Although the existence of α₂AR/OR synergy has been well characterized, its mechanism remains poorly understood. The formation of heterooligomers has been proposed as a molecular basis for interactions between neuronal G‐protein‐coupled receptors. The relevance of heterooligomer formation to spinal analgesic synergy requires demonstration of the expression of both receptors within the same neuron as well as the localization of both receptors in the same neuronal compartment. We used immunohistochemistry to investigate the spatial relationship between α₂ARs and ORs in the rat spinal cord to determine whether coexpression could be demonstrated between these receptors. We observed extensive colocalization between α_2A‐adrenergic and δ‐opioid receptors (DOP) on substance P (SP)‐immunoreactive (‐ir) varicosities in the superficial dorsal horn of the spinal cord and in peripheral nerve terminals in the skin. α_2AAR‐ and DOP‐ir elements were colocalized in subcellular structures of 0.5 μm or less in diameter in isolated nerve terminals. Furthermore, coincubation of isolated synaptosomes with α₂AR and DOP agonists resulted in a greater‐than‐additive increase in the inhibition of K⁺‐stimulated neuropeptide release. These findings suggest that coexpression of the synergistic receptor pair α_2AAR‐DOP on primary afferent nociceptive fibers may represent an anatomical substrate for analgesic synergy, perhaps as a result of protein–protein interactions such as heterooligomerization. J. Comp. Neurol. 513:385–398, 2009. © 2009 Wiley‐Liss, Inc.     

Electrophysiological investigation of β, β′-iminodipropionitrile neuropathy: Intracellular recordings in spinal cord

β, β′-Iminodipropionitrile (IDPN) was given to cats (50 mg/kg/week for 5 weeks) to induce giant axonal swellings in the proximal internodes of motor axons. Conventional intracellular recording techniques were used to investigate the influence of the axon swellings on axonal impulse conduction and generation of action potentials in unidentified lumbosacral motoneurons (MN).Action potentials recorded from axon swellings, verified by lack of orthodromically or antidromically elicited EPSPs or IPSPs, afterhyperpolarization potentials or initial segment-somaldendritic (IS-SD) inflections, were variable in shape. Some were indistinguishable from recordings in normal axons. Component or extra potentials occurred in 45% of recordings from axon swellings; their position on the action potential depended on the direction of impulse invasion into the swelling. Many action potentials were broad, with amplitudes less than 30 mV. Impulse conduction was estimated to be blocked in 19% of motor axons tested.Action potentials recorded in MN of IDPN treated cats resembled in many aspects those recorded in chromatolytic MN, with increased latencies upon antidromic stimulation and decreased IS conduction times and SD thresholds; other parameters did not differ significantly. The minimal interval between two stimuli which each evoked action potentials increased from3.3 ± 0.1to5.8 ± 0.5ms. IS-SD portions of the action potentials could not be fractionated in 49% of cells regardless of interpulse interval. Many MN failed to follow frequencies as low as 10 Hz. Delayed depolarizations were observed in 14% of MN recordings. Repetitive action potentials were elicited by single stimuli in 14% of MN and more frequently by orthodromic than antidromic stimulation. Action potentials could often be elicited in the same MN by stimulation of more than one ventral root filament. The incidence of this ephaptic transmission or crosstalk was estimated to be 12%. The findings are discussed in terms of the influence of proximal axon swellings on action potential generation in MN, propagation along non-homogeneous regions of axons and functional chromatolysis.     

Role of CNPase in the oligodendrocytic extracellular 2′,3′‐cAMP‐adenosine pathway

Extracellular adenosine 3′,5′‐cyclic monophosphate (3′,5′‐cAMP) is an endogenous source of localized adenosine production in many organs. Recent studies suggest that extracellular 2′,3′‐cAMP (positional isomer of 3′,5′‐cAMP) is also a source of adenosine, particularly in the brain in vivo post‐injury. Moreover, in vitro studies show that both microglia and astrocytes can convert extracellular 2′,3′‐cAMP to adenosine. Here, we examined the ability of primary mouse oligodendrocytes and neurons to metabolize extracellular 2′,3′‐cAMP and their respective adenosine monophosphates (2′‐AMP and 3′‐AMP). Cells were also isolated from mice deficient in 2′,3′‐cyclic nucleotide‐3′‐phosphodiesterase (CNPase). Oligodendrocytes metabolized 2′,3′‐cAMP to 2′‐AMP with 10‐fold greater efficiency than did neurons (and also more than previously examined microglia and astrocytes); whereas, the production of 3′‐AMP was minimal in both oligodendrocytes and neurons. The production of 2′‐AMP from 2′,3′‐cAMP was reduced by 65% in CNPase ?/? versus CNPase +/+ oligodendrocytes. Oligodendrocytes also converted 2′‐AMP to adenosine, and this was also attenuated in CNPase ?/? oligodendrocytes. Inhibition of classic 3′,5′‐cAMP‐3′‐phosphodiesterases with 3‐isobutyl‐1‐methylxanthine did not block metabolism of 2′,3′‐cAMP to 2′‐AMP and inhibition of classic ecto‐5′‐nucleotidase (CD73) with α,β‐methylene‐adenosine‐5′‐diphosphate did not attenuate the conversion of 2′‐AMP to adenosine. These studies demonstrate that oligodendrocytes express the extracellular 2′,3′‐cAMP‐adenosine pathway (2′,3′‐cAMP → 2′‐AMP → adenosine). This pathway is more robustly expressed in oligodendrocytes than in all other CNS cell types because CNPase is the predominant enzyme that metabolizes 2′,3′‐cAMP to 2‐AMP in CNS cells. By reducing levels of 2′,3′‐cAMP (a mitochondrial toxin) and increasing levels of adenosine (a neuroprotectant), oligodendrocytes may protect axons from injury. GLIA 2013;61:1595–1606     

Reelin and the Cdc42/Rac1 guanine nucleotide exchange factor αPIX/Arhgef6 promote dendritic Golgi translocation in hippocampal neurons

In the cerebral cortex of reeler mutant mice lacking reelin expression, neurons are malpositioned and display misoriented apical dendrites. Neuronal migration defects in reeler have been studied in great detail, but how misorientation of apical dendrites is related to reelin deficiency is poorly understood. In wild‐type mice, the Golgi apparatus transiently translocates into the developing apical dendrite of radially migrating neurons. This dendritic Golgi translocation has recently been shown to be promoted by reelin. However, the underlying signalling mechanisms are largely unknown. Here, we show that the Cdc42/Rac1 guanine nucleotide exchange factor αPIX/Arhgef6 promoted translocation of Golgi cisternae into developing dendrites of hippocampal neurons. Reelin treatment further increased the αPIX‐dependent effect. In turn, overexpression of exchange activity‐deficient αPIX or dominant‐negative (dn) Cdc42 or dn‐Rac1 impaired dendritic Golgi positioning, an effect that was not compensated by reelin treatment. Together, these data suggest that αPIX may promote dendritic Golgi translocation, as a downstream component of a reelin‐modulated signalling pathway. Finally, we found that reelin promoted the translocation of the Golgi apparatus into the dendrite that was most proximal to the reelin source. The distribution of reelin may thus contribute to the selection of the process that becomes the apical dendrite.     

Minocycline and fluorocitrate suppress spinal nociceptive signaling in intrathecal IL‐1β–induced thermal hyperalgesic rats

We previously demonstrated that intrathecal IL‐1β caused thermal hyperalgesia in rats. This study was conducted to examine the effects and cellular mechanisms of glial inhibitors on IL‐1β–induced nociception in rats. The effects of minocycline (20 μg), fluorocitrate (1 nmol), and SB203580 (5 μg) on IL‐1β (100 ng) treatment in rats were measured by nociceptive behaviors, western blotting of p38 mitogen‐activated protein kinase (MAPK) and inducible nitric oxide synthase (iNOS) expression, cerebrospinal fluid nitric oxide (NO) levels, and immunohistochemical analyses. The results demonstrated that intrathecal IL‐1β activated microglia and astrocytes, but not neurons, in the dorsal horn of the lumbar spinal cord, as evidenced by morphological changes and increased immunoreactivity, phosphorylated p38 (P‐p38) MAPK, and iNOS expression; the activation of microglia and astrocytes peaked at 30 min and lasted for 6 h. The immunoreactivities of microglia and astrocytes were significantly increased at 30 min (6.6‐ and 2.7‐fold, respectively) and 6 h (3.3‐ and 4.0‐fold, respectively) following IL‐1β injection, as compared with saline controls at 30 min (all P < 0.01). IL‐1β induced P‐p38 MAPK and iNOS expression predominantly in microglia and less in astrocytes. Minocycline, fluorocitrate, or SB203580 pretreatment suppressed this IL‐1β–upregulated P‐p38 MAPK mainly in microglia and iNOS mainly in astrocytes; minocycline exhibited the most potent effect. Minocycline and fluorocitrate pretreatment abrogated IL‐1β–induced NO release and thermal hyperalgesia in rats. In conclusion, minocycline, fluorocitrate, and SB203580 effectively suppressed the IL‐1β–induced central sensitization and hyperalgesia in rats. © 2012 Wiley Periodicals, Inc.     

α-Melanotropin and β-endorphin-like immunoreactivities are contained within neurons and nerve fibers of the rat duodenum

Both α-melanotropin and β-endorphin were revealed by immunofluorescence microscopy studies within neurons and nerve fibers of the rat duodenum. An immunohistochemical staining for α-melanotropin was seen within neuronal cell bodies and nerve fibers bundles of the myenteric and submucous plexus. A β-endorphin immunofluorescence was visualized within perikarya and nerve fibers of both the myenteric and submucous plexus. α-Melanotropin as well as β-endorphin immunoreactivities were strictly localized to structures of the enteric nervous system. In crypts and epithelial cells only a non-specific staining was observed.