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Our previous study identified a new form of spinocerebellar ataxia (SCA), in which mutations in the gene coding for transglutaminase 6 (TG6) were suggested to be causative. However, the data concerning cellular distribution of TG6 in the brain is still fragmentary. Therefore, we now report a comprehensive immunohistochemical examination of the expression profile of TG6 in adult mouse brain. TG6 was abundantly expressed in the septal region, basal ganglia, hypothalamus and brainstem. Notably, numerous TG6-positive neurons were found in the key brain regions involved in regulating locomotion activity, including the globus pallidus, subthalamic nucleus, substantia nigra, cerebellum, some precerebellar nuclei, and spinal motor neurons. Double immunostaining showed that the vast majority of TG6-positive neurons in the reticular nigra were GABAergic and those in the compact nigra were not dopaminergic. In addition, double staining for TG6 with either anti-NeuN or glial fibrillary acidic protein (GFAP) antibodies demonstrated exclusive NeuN-TG6 co-localization. This study presents a comprehensive overview of TG6 expression in the mouse brain, and provides insight for investigating the role of TG6 in the development of SCA. Anat Rec, 296:1576–1587, 2013. © 2013 Wiley Periodicals, Inc.
The spinocerebellar ataxias (SCAs) are a large clinically and genetically heterogeneous group of inherited neurodegenerative disorders that are characterized by dysfunction of the cerebellum, as shown by progressive loss of balance and motor coordination of gait and limbs (Duenas et al., 2006). Atrophies of the cerebellum and brainstem are the prominent features, but different combinations of degeneration in the cerebellum, spinal tracts, peripheral nerves, cerebral cortex, basal ganglia, optic nerve, and others are also observed in SCA (Durr, 2010). Several cellular and molecular mechanisms have been proposed to be critical for the development of SCAs, such as toxic accumulation of aggregates and intranuclear inclusions, transcription interference, altered calcium homoeostasis, altered glutamate transmission, mitochondrial dysfunction, RNA alterations, chromatin structure abnormalities, toxic accumulation of aggregates and RNA foci, cytoskeletal abnormalities, axonal transport deficits, and increased tau phosphorylation (Durr, 2010). In 2010, our group identified two missense mutations in the TGM6 gene in two Chinese SCA families, c.1550T>G transition (p.L517W) and c.980A>G transition (p.D327G), suggesting a novel causative gene for SCA, named as SCA 35 (Wang et al., 2010). In 2012, Li et al. (Li et al., 2013) reported a novel mutation in the TGM6 gene in another Chinese SCA family, c. 1528G>C transition (p.D510H), which was supportive of the TGM6 gene being causative gene for SCA 35. In addition, overexpression of the two missense mutations of TGM6 (D327G, L517W) dramatically increases the sensitivity of NIH3T3 cells to staurosporine-induced apoptosis via increasing the activity of caspases (Guan et al., 2013).
Transglutaminases (TGMs) form a family of structurally- and functionally related enzymes that post-translationally modify proteins by catalyzing a Ca2+-dependent transferase reaction between the c-carboxamide group of a peptide-bound glutamine residue and various primary amines (Yee et al., 1994). Nine distinctly expressed TGM genes are present in mammals (Fesus and Piacentini, 2002). Among them, TG 1–3 and 6 were discovered in human brain (Hadjivassiliou et al., 2008). In addition to SCA, TGs are also hypothesized to be involved in the pathogenesis of several other neurodegenerative diseases, including polyglutamine expansion diseases, Alzheimer's, Parkinson's and supranuclear palsy (Jeitner et al., 2009). However, despite extensive investigation over the last two decades, the physiologic or pathologic roles of TGs in the brain remain unclear.
Although the functions of the TG6 protein implicated in SCA are presently unknown, analysis of its expression pattern in the mammalian brain could facilitate our understanding of the mechanisms that underlie the neuropathology of the disease. Therefore, in this study we investigated the distribution of TG6 in adult mouse brain by immunohistochemistry and found that a large number of TG6-positive neurons were located in brain regions regulating locomotor activity.
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A previous study has investigated TG6 expression in mouse central nervous system at embryonic stages, and identified an abundant expression of TG6 adult brain (Thomas et al., 2013). Overall, our results were consistent with this report, and we provided comprehensive immunohistochemical mapping data throughout the whole brain regions. Robust TG6 expression in brain regions involved in locomotive control, such as the globus pallidus, subthalamic nucleus, sustantia nigra, ventricular nucleus, deep cerebellar nucleus, supports the hypothesis that TG6 is associated with the neurodegenerative disease and TG6 is the causative gene for SCA 35 (Wang et al., 2010).
Our previous study showed that walking difficulty and cerebellar dysarthria were early clinical signs, while ataxia of the upper extremities was a relatively late symptom in SCA 35 families (Wang et al., 2010). The onset age ranged from 40 to 48 years and disease duration varied from 5 to 31 years (Wang et al., 2010). In the report by Li et al. (2013), patients presented with similar symptoms to those reported by our patients, including unsteady gait, spasticity, ataxia, hyperreflexia, and Babinski's sign, while the onset age of the patients ranged from childhood to 40s. These clinical symptoms are more likely related to extracerebellar pathology because imaging and neuropathological data show that major alterations are present in other parts of the motor system outside the cerebellum in SCA (van Gaalen et al., 2011).
Consistent with the clinical observations from SCA patients, TG6-positive neurons were abundantly distributed in both the cerebellum and other brain regions involved in motor control in this study. In the cerebellum, TG6-positive neurons were present in both the cerebellar cortex and cerebellar nuclei. In the cerebellar cortex, TG6 was only expressed in Purkinje cells, which are the only neurons responsible for sending output from the cortex. On the other hand, TG6 was expressed throughout the deep cerebellar nuclei which receive input from Purkinje cells and send output information to other brain regions. In addition, numerous TG6-positive neurons were contained in the pontine nucleus and vestibular nucleus, which send many mossy fiber axons to the cerebellum (Coesmans et al., 2004). The inferior olivary nucleus, which sends climbing fibers to the cerebellum (Devor, 2000), also contained TG6-positve neurons. In the view of direct efferent targets of the cerebellum, many TG6-positive neurons were located in the red nucleus and tectum. Taken together, TG6 was expressed in the cerebellum and those brain regions with direct neuronal connections with the cerebellum.
In addition to the globus pallidus and caudate putamen, the ventral pallidus, substantia innominata, substantia nigra, and subthalamic nucleus are also regarded as a part of the basal ganglia in terms of their functions in motor control. These regions, together with the cerebellum, are regarded as the key components of the extrapyramidal system (Bostan and Strick, 2010). Interestingly, in this study, except for the caudate putamen, TG6 was expressed in numerous neurons in these regions. Mutations of the TGM6 gene may lead to functional impairment and finally neuronal loss in these brain regions as SCA development progresses. An in vivo study has shown that intraventricular injection of anti-TG2/3/6 cross-reactive antibody provoked ataxia in mice (Boscolo et al., 2010). Taken together, the high distribution density of TG6-positive neurons in the extrapyramidal system supports the hypothesis that TGM6 is the causative gene in the development of SCA 35 (Wang et al., 2010).
Double immunostaining showed that TG6 was expressed in the reticular part of the sustantia nigra and reticular thalamic nucleus, which are the two brain regions containing a high density of GABAergic neurons (Brazhnik et al., 2008). GABA is the main inhibitory neurotransmitter in the brain. Our group has reported that the inhibitory amino acids, including GABA, in the cerebrospinal fluid of SCA patients is reduced (Yang et al., 1999), which may reflect an impairment or loss of GABAergic neurons in SCA patients.
Recently, several reports showed TG enzymes are also related to other neurodegenerative diseases. It has been reported that TG activity is significantly elevated in the affected cerebral regions in Alzheimer disease, Huntington disease and supranuclear palsy (Jeitner et al., 2009). In addition, increased TG2 protein is observed in the cerebrospinal fluid of Alzheimer disease (Bonelli et al., 2002) and Parkinson disease subjects (Vermes et al., 2004). It is likely that in addition to SCA TG6 may be also involved in the development of other neurodegenerative diseases.
In summary, we have described the detailed TG6 expression pattern in adult mouse CNS for the first time, and our data provide a morphological basis for studying the role of TG6 in the nervous system and neurodegenerative disorders, especially for exploring the pathogenesis of SCA35 in the future.