Human neural stem cells over-expressing choline acetyltransferase restore cognition in rat model of cognitive dysfunction.

A human neural stem cell (NSC) line over-expressing human choline acetyltransferase (ChAT) gene was generated and these F3.ChAT NSCs were transplanted into the brain of rat Alzheimer disease (AD) model which was induced by application of ethylcholine mustard aziridinium ion (AF64A) that specifically denatures cholinergic nerves and thereby leads to memory deficit as a salient feature of AD. Transplantation of F3.ChAT human NSCs fully recovered the learning and memory function of AF64A animals, and induced elevated levels of acetylcholine (ACh) in cerebrospinal fluid (CSF). Transplanted F3.ChAT human NSCs were found to migrate to various brain regions including cerebral cortex, hippocampus, striatum and septum, and differentiated into neurons and astrocytes. The present study demonstrates that brain transplantation of human NSCs over-expressing ChAT ameliorates complex learning and memory deficits in AF64A-cholinotoxin-induced AD rat model.

Human neural stem cells overexpressing choline acetyltransferase restore cognitive function of kainic acid-induced learning and memory deficit animals.

Alzheimer disease (AD) is a progressive neurodegenerative disease, which is characterized by loss of memory and cognitive function. In AD patients dysfunction of the cholinergic system is the main cause of cognitive disorders, and decreased activity of choline acetyltransferase (ChAT), an enzyme responsible for acetylcholine (ACh) synthesis, is observed. In the present study we investigated if brain transplantation of human neural stem cells (NSCs) genetically modified to encode ChAT gene improves cognitive function of kainic acid (KA)-induced learning deficit rats. Intrahippocampal injection of KA to hippocampal CA3 region caused severe neuronal loss, resulting in profound learning and memory deficit. F3.ChAT human NSCs transplanted intracerebroventricularly improved fully the learning and memory function of KA-induced learning deficit animals, in parallel with the elevation of ACh levels in cerebrospinal fluid. F3.ChAT human NSCs migrated to the KA-induced injury site (CA3) and differentiated into neurons and astrocytes. The present study demonstrates that human NSCs expressing ChAT have lesion-tropic property and improve cognitive function of learning deficit model rats with hippocampal injury by increasing ACh level.

Nuclear choline acetyltransferase activates transcription of a high-affinity choline transporter.

Choline acetyltransferase (ChAT) synthesizes the neurotransmitter, acetylcholine, at cholinergic nerve terminals. ChAT contains nuclear localization signals and is also localized in the nuclei of neural and non-neuronal cells. Nuclear ChAT might have an as yet unidentified function, such as transcriptional regulation. In this study, we investigated the alteration of candidate gene transcription by ChAT. We chose high affinity choline transporter (CHT1) and vesicular acetylcholine transporter (VACHT) as candidate genes, which function together with ChAT in acetylcholine production. Using SH-SY5Y human neuroblastoma cells stably expressing wild-type human ChAT, we found that overexpressed ChAT enhanced transcription of the CHT1 gene but not the VACHT gene. In contrast, nuclear localization signal disrupted, and catalytically inactive mutant ChATs could not induce, CHT1 expression. Additionally, ChAT did not alter CHT1 expression in non-neuronal HEK293 cells. Our results suggest that ChAT activates the transcription of selected target genes in neuronal cells. Both enzymatic activity and nuclear translocation of ChAT are required for its transcriptional enhancement.

Human CRB2 inhibits gamma-secretase cleavage of amyloid precursor protein by binding to the presenilin complex.

Drosophila Crumbs has been reported to attenuate Notch signaling by inhibition of gamma-secretase cleavage at the wing margins. gamma-Secretase is an intramembrane protease that is responsible for the generation of amyloid-beta (Abeta) peptides from the beta-amyloid precursor protein (APP). Here, we re-examined gamma-secretase inhibition by human CRB2, which is the most abundant Crumbs ortholog in the brain. Transfected CRB2 inhibited proteolytic production of Abeta and APP intracellular domains from APP C-terminal fragments in HEK293 and SH-SY5Y cells. Conversely, knockdown of endogenous CRB2 increased gamma-secretase cleavage products in SH-SY5Y cells. CRB2 inhibition of gamma-cleavage was also detected in cell-free assays. CRB2 interacted with the gamma-secretase complex, but was not a competitive substrate for gamma-cleavage. The transmembrane domain of CRB2 was indispensable for inhibition of Abeta generation and mediated CRB2 binding with the gamma-secretase complex. In addition, the cytoplasmic domain appeared to play a supportive role in gamma-secretase inhibition, whereas mutational disruption of the two protein-binding motifs involved in the formation of cell adhesion complexes did not affect gamma-secretase inhibition. Co-overexpression of presenilin-1 or APH-1 abrogated gamma-secretase inhibition probably through prevention of the incorporation of CRB2 into the gamma-secretase complex. Our results suggest that CRB2 functions as an inhibitory binding protein that is involved in the formation of a mature but inactive pool of the gamma-secretase complex.

Axotomy alters alternative splicing of choline acetyltransferase in the rat dorsal motor nucleus of the vagus nerve.

Choline acetyltransferase of the peripheral type (pChAT) is a splice variant that lacks exons 6-9 of the common-type ChAT (cChAT); the role of pChAT remains unknown. We investigated the expression of pChAT and cChAT after axotomy to try to elucidate its function. In the dorsal motor nucleus of the vagus nerve (DMNV), nucleus ambiguus (NA), and hypoglossal nucleus (HN) of control rats, we observed neural expression of cChAT but no pChAT-positive neurons. Following nerve transection, we clearly detected pChAT-labeled neurons in the DMNV and weakly labeled neurons in the NA, but pChAT was not seen in the HN. In the DMNV, the mean number of cChAT-positive neurons decreased rapidly to 40.5% of control at 3 days post transection, and to 5.0% of control after 7 days. The number of cChAT-positive neurons then gradually increased and reached a plateau of about 25% of control value at 28 days post transection. pChAT-positive neurons did not appear until 7 days after transection. On the same day, pChAT mRNA was detected in the DMNV neurons by reverse transcription-polymerase chain reaction (RT-PCR) by using laser capture microdissection. The number of pChAT-positive neurons gradually decreased, and only 10% of the cholinergic neurons retained pChAT expression 56 days post transection. Double-immunofluorescence analysis showed that some of the DMNV neurons expressed both cChAT and pChAT upon recovery from axotomy. These results suggest that the expression of pChAT is associated with the regenerative or degenerative processes of motoneurons especially for general visceral efferents.

First visualization of cholinergic cells and fibers by immunohistochemistry for choline acetyltransferase of the common type in the optic lobe and peduncle complex of Octopus vulgaris.

This study provides the first immunohistochemical evidence visualizing cholinergic octopus neurons containing choline acetyltransferase (ChAT), the synthetic enzyme of acetylcholine. Because the antiserum applied here was raised against a recombinant protein encoded by exons 7 and 8 of the rat gene for ChAT, and initially used for studies in mammals, to validate antibody specificity for the octopus counterpart enzyme we therefore used three methods. Immunoprecipitation using Pansorbin indicated that immunoreactive octopus brain molecules were capable of synthesizing acetylcholine. Western blot analysis after denatured gel electrophoresis of octopus brain extracts revealed a single band at approximately 81 kDa. A gel slice containing the 81-kDa protein after native (nondenatured) gel electrophoresis exhibited high ChAT activity. All findings obtained with these three methods clearly indicated that the antiserum effectively recognizes octopus ChAT. The immunohistochemical use of the antiserum in the retina, optic lobe, and its neighboring peduncle complex detected enzyme-containing neuronal cell bodies in only two regions, the cell islands of the optic lobe medulla and the cortical layer of the posterior olfactory lobule. Immunoreactive fibers and probable nerve terminals were also found in the plexiform layer of the deep retina, within the stroma of the optic gland, and the neuropils of the optic lobe, peduncle lobe, and olfactory lobe. These results provide information on the morphology and distribution patterns of cholinergic neurons in the octopus visual system, a useful invertebrate model for learning and memory where the cholinergic system, as in higher vertebrates including mammals, plays an important role.

Distribution of a splice variant of choline acetyltransferase in the trigeminal ganglion and brainstem of the rat: comparison with calcitonin gene-related peptide and substance P.

Rat trigeminal ganglion neurons have been shown to contain a splice variant of choline acetyltransferase (pChAT). Here we report the distribution pattern of pChAT-containing afferents from the trigeminal ganglion to the brainstem, compared with that of calcitonin gene-related peptide (CGRP) and substance P (SP), by use of the immunohistochemical techniques in the rat. Most of CGRP(+) SP(+) ganglion cells contain pChAT, whereas half of the pChAT(+) ganglion cells possess neither CGRP nor SP. In the brainstem, pChAT(+) nerve fibers are found exclusively in the trigeminal and solitary systems, although the distribution pattern differs from that of CGRP(+) or SP(+) fibers. First, the ventral portion of the principal sensory nucleus contains many pChAT(+) fibers, with few CGRP(+) or SP(+) fibers. Because this portion receives projections of nociceptive corneal afferents, a subpopulation of pChAT(+) CGRP(-) SP(-) primary afferents is most probably nonpeptidergic nociceptors innervating the cornea. Second, the superficial laminae of the medullary dorsal horn, the main target of nociceptive afferents, contain dense CGRP(+) and SP(+) fibers but sparse pChAT(+) fibers. Because pChAT occurs in most CGRP(+) SP(+) ganglion cells, such sparseness of pChAT(+) fibers implies poor transportation of pChAT to axon branchlets. Another important finding is that pChAT(+) axons are smooth and nonvaricose, whereas CGRP(+) or SP(+) fibers possess numerous varicosities. Our confocal microscopy suggests colocalization of these three markers in the same single axons in some brainstem regions. The difference in morphological appearance, nonvaricose or varicose, appears to reflect the difference in intraaxonal distribution between pChAT and CGRP or SP.

The endoplasmic reticulum-Golgi pathway is a target for translocation and aggregation of mutant superoxide dismutase linked to ALS.

Mutations in superoxide dismutase 1 (SOD1) are responsible for 20% cases of familial amyotrophic lateral sclerosis (ALS). However, the mechanism of motor neuron degeneration caused by ALS-linked SOD1 mutants is not fully understood. Here, we used novel live cell imaging techniques to demonstrate the subcellular localization of EGFP-fused SOD1 of both wild-type (WT) and ALS-linked mutant forms in the endoplasmic reticulum (ER) and Golgi. The presence of WT and mutant SOD1 species in luminal structures was further confirmed by immunoblotting analysis of microsomal fractions from spinal cord lysates of SOD1 transgenic mice prepared by sucrose density-gradient ultracentrifugation. Chemical cross-linking studies also revealed an age-dependent aggregation of mutant SOD1, but not of WT SOD1, prominently in the microsomal fraction. Cell-free translocation assays provided evidence that monomeric SOD1 is a molecular form that can be translocated into luminal structures in the presence of ATP. Our finding that the ER-Golgi pathway is a predominant cellular site of aggregation of mutant SOD1 suggests that secretion could play a key role in pathogenesis, which is in line with the view that the disease is non-cell autonomous.

The production of antibodies that distinguish rat choline acetyltransferase from its splice variant product of a peripheral type.

To produce antibodies that permit the immunohistochemical discrimination of choline acetyltransferase of the common type (cChAT) from its splice variant of a peripheral type (pChAT), we immunized rabbits with a cChAT specific recombinant protein encoded by ChAT exons 7 and 8 of the rat cChAT gene. Successful antibody production was proved by Western blotting on rat brain and on HEK293 cells expressing green fluorescent protein (GFP), cChAT-GFP and pChAT-GFP. By immunohistochemistry our antiserum clearly labeled known cholinergic structures in rat brain, but gave no positive staining in the trigeminal ganglion which contained many neurons positive with pChAT antiserum.

Demonstration of choline acetyltransferase of a peripheral type in the rat heart.

Cholinergic innervation of the heart has been analyzed using cholinergic markers including acetylcholinesterase, choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT). In the present study we demonstrate putative cholinergic nerves in the rat heart using an antibody to ChAT of a peripheral type (pChAT), which is the product of a splice variant of ChAT mRNA and preferentially localized to peripheral cholinergic nerves. Expression of mRNAs for pChAT and the conventional form of ChAT (cChAT) were verified in the rat atrium by RT-PCR. Localization of both protein products in the atrium was confirmed by Western blotting. Virtually all neurons and small intensely fluorescent cells in the intrinsic cardiac ganglia were stained immunohistochemically for pChAT. The density of pChAT-positive fibers was very high in the conducting system, high in both atria, the right atrium in particular, and low in the ventricular walls. pChAT and VAChT immunoreactivities were closely associated in some fibers and fiber bundles in the ventricular walls. These results indicate that intrinsic cardiac neurons homogeneously express both pChAT and cChAT. Furthermore, innervation of the ventricular walls by pChAT- and VAChT-positive fibers provides morphological evidence for a significant role of cholinergic mechanisms in ventricular functions.

Rat choline acetyltransferase of the peripheral type differs from that of the common type in intracellular translocation.

Choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, has been implicated to involve multiple isoforms of ChAT mRNA in several animals. Since these isoforms are mostly non-coding splice variants, only a homologous ChAT protein of about 68 kDa has been shown to be produced in vivo. Recent evidence indicates the existence of a protein coding splice variant of ChAT mRNA, which lacks exons 6-9 of the rat ChAT gene. The encoded protein was designated ChAT of a peripheral type (pChAT), because of its preferential expression in the peripheral nervous system as confirmed by Western blot and immunohistochemistry. However, functional significance of pChAT is unknown. To obtain a clue to this question, we examined a possible difference in intracellular trafficking between pChAT and the well-known ChAT of the common type (cChAT) using green fluorescent protein (GFP) in living human embryonic kidney cells. Confocal laser scanning microscopy revealed that pChAT-GFP was detectable in the cytoplasm but not in the nucleus, whereas cChAT-GFP was found in both cytoplasm and nucleus. Following treatment with leptomycin B, a nuclear export pathway inhibitor, pChAT-GFP became detectable in both cytoplasm and nucleus, indicating that pChAT can be translocated to the nucleus. In contrast, the leptomycin B treatment did not seem to affect the content of intranuclear cChAT-GFP. After incubation with protein kinase C inhibitors, enhanced accumulation of pChAT-GFP but not cChAT-GFP occurred in the nucleus. These results clearly indicate that pChAT varies from cChAT in intracellular transportation, probably reflecting the difference in physiological roles between pChAT and cChAT.

Topographical and cytopathological lesion analysis of the white matter in Binswanger's disease brains.

The purpose of the present study was to document the topographical and cytopathological lesions in the white matter (WM) of Binswanger's disease (BD) brains. Subcortical WM lesions in each lobe and fiber bundle lesions related to the medial thalamic and hippocampal structures in clinicopathologically proven BD brains were evaluated by Klüver-Barrera staining using a grading score. Lesions in the frontal subcortical WM of BD brains, brains from non-neurological patients, and brains with cerebral hemorrhage or large cortical infarcts were also examined immunohistochemically using molecular markers for axonal flow damage: amyloid precursor protein (APP); and for demyelinating axonopathy: encephalitogenic peptide (EP). Our results indicated that the WM lesions in BD were significantly more prominent in the frontal periventricular and subcortical regions as compared with other subcortical WM lesions, in the order of the parietal, occipital and temporal lobes. Fiber bundle lesions in the capsular genu, including the anterior thalamic peduncle, were also significantly more prominent in BD brains as compared with the other bundle lesions. Furthermore, the frequency of damaged nerve fibers labeled by the EP antiserum and APP immunoreactive fibers was significantly higher in BD brains as compared with the control brains. The grading scores for the WM damage correlated significantly with those for the APP and EP immunoreactive fibers in all brains, including the control brains. The axonal damage in the frontal WM lesions of the BD brains was clearly revealed in our study using immunohistochemistry for APP and EP.

Different mechanisms of corpus callosum atrophy in Alzheimer's disease and vascular dementia.

Previous neuroimaging studies have indicated that corpus callosum atrophy in Alzheimer's disease (AD) and large vessel occlusive disease (LVOD) is caused by interhemispheric disconnection, namely Wallerian degeneration of interhemispheric commissural nerve fibers originating from pyramidal neurons in the cerebral cortex. However, this hypothesis has not been tested from a neuropathological viewpoint. In the present study, 22 brains with AD (presenile onset, 9; senile onset, 13), 6 brains with Binswanger's disease (BD), a form of vascular dementia and 3 brains with LVOD were compared with 6 non-neurological control brains. White matter lesions in the deep white matter and corpus callosum were quantified as a fiber density score by image analysis of myelin-stained sections. Axonal damage and astrogliosis were assessed by immunohistochemistry for amyloid precursor protein and glial fibrillary acidic protein, respectively.The corpus callosum thickness at the anterior part of the body was decreased in AD and LVOD,but not in BD significantly, as compared with the controls. The corpus callosum thickness correlated roughly with brain weight in AD (R=0.50),and with the severity of deep white matter lesions in BD (R=0.81). Atrophy of the brain and corpus callosum was more marked in presenile onset AD than in senile onset AD. With immunohistochemistry, the corpus callosum showed axonal damage and gliosis with a decreased fiber density score in BD and LVOD, but not in AD. Thus, corpus callosum atrophy was correlated with brain atrophy in AD, which is relevant to the mechanism of interhemispheric disconnection,whereas corpus callosum lesions in BD were secondary to deep white matter lesions. Corpus callosum atrophy in LVOD may indicate interhemispheric disconnection, but focal ischemic injuries may also be involved.

Innervation of rat iris by trigeminal and ciliary neurons expressing pChAT, a novel splice variant of choline acetyltransferase.

We have recently discovered a splice variant of choline acetyltransferase (ChAT) mRNA and designated the variant protein pChAT because of its preferential expression in peripheral neuronal structures. In this study, the presence of pChAT in rat iris was examined by immunohistochemistry and Western blot using a pChAT antiserum, in combination with RT-PCR analysis and ChAT enzyme assay. For comparison, the conventional ChAT (cChAT) was studied in parallel. By pChAT immunohistochemistry, intense labeling was found to occur in nerve fibers of the iris and in neurons of the ciliary and trigeminal ganglia. Denervation studies, analyzed by semiquantitative morphometry, indicated that these iridial pChAT fibers originated about half from the ciliary ganglion and the other half from the trigeminal ganglion. The presence of pChAT protein in the iris and trigeminal ganglion was confirmed by Western blot. The expression of pChAT mRNA in the ciliary and trigeminal ganglia was proved by RT-PCR. Although cChAT protein and mRNA were detected in the ciliary ganglion, neither was detectable in the trigeminal ganglion. The contributions of the ciliary and trigeminal ganglia to the iridial ChAT enzyme activity were verified by the present ChAT assay. Here, we provide evidence that iridial pChAT nerves are composed of postganglionic parasympathetic efferents from the ciliary ganglion and, more interestingly, somatic sensory afferents of the trigeminal ophthalmic nerve.

Demonstration of cholinergic ganglion cells in rat retina: expression of an alternative splice variant of choline acetyltransferase.

Acetylcholine acts as a neurotransmitter in the retina. Although previous physiological studies have indicated that some retinal ganglion cells may be cholinergic, several immunohistochemical studies using antibodies to choline acetyltransferase (ChAT) have stained only amacrine cells but not ganglion cells. Recently, we identified a splice variant of ChAT mRNA, lacking exons 6-9, in rat peripheral nervous system. The encoded protein was designated as ChAT of a peripheral type (pChAT), against which an antiserum was raised. In the present study, we examined expression of pChAT in rat retina, both at the protein level by immunohistochemistry using the antiserum and at the mRNA level by RT-PCR. Immunohistochemistry revealed that although no positive neurons were found in untreated intact retinas, many neurons became immunoreactive for pChAT after intravitreal injection of colchicine. Damage of the optic nerve was also effective in disclosing positive cells. Such positive neurons were shown to be ganglion cells by double labeling with a retrograde tracer that had been injected into the contralateral superior colliculus. Western blot analysis and RT-PCR revealed a corresponding band to the pChAT protein and to the amplified pChAT gene fragment, respectively, in retinal samples. In addition, ChAT activity was definitely detected in retinofugal fibers of the optic nerve. These results indicate the presence of cholinergic ganglion cells in rat retina.

Axonal damage and demyelination in the white matter after chronic cerebral hypoperfusion in the rat.

Cerebral white matter (WM) lesions are observed frequently in human ischemic cerebrovascular disease and have been thought to contribute to cognitive impairment. This type of lesion can be experimentally induced in rat brains under chronic cerebral hypoperfusion by the permanent occlusion of both common carotid arteries. However, it remains uncertain whether chronic ischemia can damage both the gray and white matter, and whether it can induce demyelination with or without axonal damage. Therefore, we examined axonal damage using immunohistochemistry for the amyloid beta/A4 precursor protein (APP), chromogranin A (CgA) and demyelination using immunohistochemistry for the encephalitogenic peptide (EP) in this model. Severe WM lesions such as vacuolation and the loss of nerve fibers appeared in the optic nerve and optic tract after 3 days of ligation, and less intense changes were observed in the corpus callosum, internal capsule, and fiber bundles of the caudoputamen after 7 days with Klüver-Barrera and Bielschowsky staining. These WM lesions persisted even after 30 days. The APP, CgA, and EP-immunopositive fibers increased in number from 1 to 30 days after the ligation in the following WM regions: the optic nerve, optic tract, corpus callosum, internal capsule, and fiber bundles of the caudoputamen. In contrast, only a few APP, CgA, or EP-immunopositive fibers were detected in the gray matter regions, including the cerebral cortex and hippocampus. These results indicate that the WM is more susceptible to chronic cerebral hypoperfusion than the gray matter, with an involvement of both axonal and myelin components. Furthermore, immunohistochemistry for APP, CgA, and EP is far superior to routine histological staining in sensitivity and may become a useful tool to investigate WM lesions caused by various pathoetiologies.