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Death Receptor 3 is a proinflammatory member of the immunomodulatory tumour necrosis factor receptor superfamily, which has been implicated in several inflammatory diseases such as arthritis and inflammatory bowel disease. Intriguingly however, constitutive DR3 expression has been detected in the brains of mice, rats and humans although its neurological function remains unknown. By mapping the normal brain expression pattern of DR3, we found that DR3 is expressed specifically by cells of the neuron lineage in a developmentally regulated and region specific pattern. Behavioural studies on DR3-deficient (DR3ko) mice showed that constitutive neuronal DR3 expression was required for stable motor control function in the aging adult. DR3ko mice progressively developed behavioral defects characterised by altered gait, dyskinesia, and hyperactivity, which were associated with elevated dopamine and lower serotonin levels in the striatum. Importantly, retrograde tracing showed that absence of DR3 expression led to the loss of corticostriatal innervation without significant neuronal loss in aged DR3ko mice. These studies indicate that DR3 plays a key non-redundant role in the retention of normal motor control function during aging in mice and implicate DR3 in progressive neurological disease.
The process of aging in the brain is complex and characterised by progressive alterations to neural signalling pathways affecting behavior (O'Sullivan et al., 2001; Andrews-Hanna et al., 2007). Age-related declines in motor control function appear to reflect environmental and genetic components altering neuron lifespan and function (Mattson et al., 2002; Heuninckx et al., 2005; Yankner et al., 2008). Continued motor control function and spatial awareness are critically reliant upon neuronal signalling between the cortex, which translates sensory input, and the striatum (Goldman-Rakic, 1987; Rakic, 1988), which mediates motor and cognitive processes (Goldman-Rakic, 1987; Dube et al., 1988; Rakic, 1988; Albin et al., 1989; Smith et al., 1994; Groenewegen, 2003; Grahn et al., 2008). The neurotransmitter dopamine plays a primary role in the conduction of all motor control signalling within the striatum (Di Matteo et al., 2008; Palmiter, 2008; Yao et al., 2008). The processes that govern the homeostatic maintenance of these neural pathways following their formation in the aging adult are poorly understood.
Death Receptor 3 (DR3, Wsl-1, Apo3, LARD, TRAMP, TNFRSF25, TR3) (Chinnaiyan et al., 1996; Kitson et al., 1996; Marsters et al., 1996; Bodmer et al., 1997; Screaton et al., 1997) is a member of the tumor necrosis factor receptor superfamily (TNFRSF), a group of structurally related immunomodulatory cytokine receptors capable of triggering NF-κB induction and/or caspase activation (Ashkenazi and Dixit, 1998; Mackay and Kalled, 2002). DR3 along with its ligand TNF-like factor 1A (TL1A) has been described as pro-inflammatory in several murine disease models (Al-Lamki et al., 2003; Bamias et al., 2006; Bull et al., 2008; Fang et al., 2008; Meylan et al., 2008; Pappu et al., 2008; Takedatsu et al., 2008) and in chronic human diseases such as inflammatory bowel disease and rheumatoid arthritis (Osawa et al., 2004; Papadakis et al., 2005; Bamias et al., 2006; Borysenko et al., 2006; Cassatella et al., 2007). In the brain, increased DR3 expression has been reported in ischemic rats (Harrison et al., 2000), and human Alzheimer' disease (Newman et al., 2000), suggesting this receptor contributes to neuropathology. Intriguingly however, endogenous DR3 expression is also observed in the brain of normal mice, rats and humans (Marsters et al., 1996; Wang et al., 2001a; O'Keeffe et al., 2008). Furthermore, such basal expression may be neurologically important as some TNFRSF members appear to facilitate normal neuronal differentiation and survival (Kojima et al., 2000; Nieoullon and Coquerel, 2003; Graybiel, 2005; Ishiguro et al., 2007; O'Keeffe et al., 2008). Collectively, these observations suggest basal DR3 brain expression may play an important role in normal neuronal function.
In this study, we addressed the physiological role of DR3 in the adult brain by examining the behavior of DR3ko mice during aging. We report that adult DR3ko mice spontaneously develop severe behavioral defects characterised by a progressive decline in higher motor control functions with advancing age. Importantly, adult neuronal DR3 expression was found to be critical for continued maintenance of striatal neurotransmitter expression and for stable neuronal contact between the cortex and the striatum.
DR3ko mice on a C57BL/6 background were previously generated at, and colony founding animals supplied by, Cancer Research, UK (Wang et al., 2001b). All UK-based animal experiments were subject to local ethical review and conducted according to personal, project and institutional licences under the UK Animals (Scientific Procedures) Act 1986. For experiments carried out in Spain, animal-related procedures were in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and approved by the local animal care committee of the Universitat de Barcelona and by the Generalitat (Autonomous Government) of Catalonia.
Tissue preparation, TH and NeuN immunohistochemistry on free floating sections and subsequent morphometry was performed as described elsewhere (Kelly et al., 2007). Brain region volume and cell number was based on NeuN/TH staining and determined using the Olympus CASTgrid stereology system on a 1:12 series using previously described methods. At high magnification, brain region volume, neurone number and the density of TH immunostaining was measured on 4 anatomically matched sections per animal using the ×40 objective (Leica DMRB microscope (Leica Microsystems, Wetzlar, Germany)) and Scion-image system (Scion Corporation, Frederick, MD, USA).
Indirect fluorescent immunocytochemistry was performed using standard protocols with primary antibodies directed against neuronal nuclei (NeuN, Pharmingen), β-galactosidase (β-Gal, Chemicon) and glial fibrillary acidic protein (GFAP, DAKO). Sections were counter stained with Hoechst 33342 (2 μg/ml; Sigma) to label nuclei before visualization by confocal microscopy on an Olympus IX70 microscope linked into an Ultraview® system (Perkin and Elmer).
Mice were generated by replacement of the DR3 gene with a cassette including an internal ribosome entry site (IRES) and lacZ poly(A), allowing visualization using X-gal staining. Brains from DR3het and DR3ko mice were removed, flash frozen in LN2, coronally sectioned at 10 μm thickness and stored at −80°C. Whole mouse brains being assayed for β-Gal were removed, fixed in 4% PFA and then washed extensively in PBS. Sections being assayed for β-Gal were prepared as described above, immersed in X-gal staining solution for 18-24 hours at 37°C, counterstained with Nuclear Fast Red (Vector laboratories) and hard mounted using standard techniques. Images were taken using a Nikon light microscope, Kodak EPY-64T colour slide film or a Nikon based digital camera imaging system.
DR3 and TL1A expression in the brains of 8-12 weeks old mice was examined by RT-PCR. Brains from 8-12 week old mice were micro-dissected on ice into the hippocampus, basal ganglia, cortex, cerebellum, dentate gyrus and colliculi. Total RNA was extracted from tissues or cultured cells using RNeasy (Qiagen) and cDNA prepared using Superscript® II (Invitrogen) according to manufacturers instructions. PCR was carried out according to the following cycle preceded with Taq activation for 15 min at 95°C: step1, 94°C for 45 sec, an annealing step (temperature dependent upon primers) for 30 s, and 72°C for 45 sec. The reaction was terminated by 10 min at 72°C and then kept at 4°C until required. PCR primers, annealing temperature and expected product size for the amplification of TNFRSF members and TL1A are shown in Supplemental Table 1. The amount of cDNA in each PCR reaction was assessed by additionally amplifying a fragment of the GAPDH cDNA. Reaction products were resolved on a 1.2% agarose gel and stained with ethidium bromide prior to UV visualisation. Band intensities were measured using Labworks® analysis software.
Gait and balance analysis was performed using footprint pattern analysis as previously described in the presence or absence of a 1cm rod balance beam elevated by 3 cm (Dunnett et al., 1998). Open field testing was preformed as previously described (Torres et al., 2007). Open field analysis was conducted on scaled drawings of animal movement produced by frame-to-frame examination of captured digital footage. Crossovers were determined by counting the combined number of times mice crossed their own paths during observation per min during a 10 min observation period.
Male DR3het and DR3ko mice of 3- and 18-months age were sacrificed and their brains removed and micro-dissected on ice. Brain tissues were weighed, snap frozen using LN2 and sent for commercial analysis (RenaSci Consultancy LTD, UK) by HPLC to determine the level of 5-hydroxyindoleacetic acid (5-HIAA), serotonin (5-HT), 3, 4-dihydroxyphenylacetic acid (DOPAC), dopamine, homovanillic acid (HVA) and noradrenaline (NA).
Intracerebral injection of the retrograde tracer, Fluorogold (FG), (Fluorochrome Inc. Denver, CO, USA) was performed in 3 and 18 month-old mice as previously described (Pineda et al., 2005). In brief, animals were anaesthetized, placed in a stereotaxic apparatus (Stoelting Co., Wood Dale, IL, USA) and Fluorogold solution (1% in PBS) was injected into the striatum at the following coordinates: A/P +0.5 mm, L −2 mm from bregma; and D/V 2.7 mm from dura; incisor bar set at −3 mm. After 2 days, mice were perfused transcardially with 4% paraformaldehyde in PBS, cryoprotected with 30% sucrose and frozen in dry-ice cooled isopentane. Coronal sections (30 μm) were cut on a cryostat and mounted with Mowiol (Calbiochem, San Diego, CA, USA).
Statistical analyses were made with either Student t-Tests or t-Tests assuming unequal variance if normal distributions could not be assumed. A Chi-squared test was used to compare frequency of run completion data. Unless stated otherwise, plotted data represent mean ± sem; p values of <0.05 were considered significant.
Analysis of DR3 mRNA transcript levels in mouse (Wang et al., 2001a; O'Keeffe et al., 2008) and rat (Harrison et al., 2000) tissue has previously shown that DR3 is expressed using alternate length transcripts in the brain compared to lymphoid tissue. In the absence of a range of antibodies that are confirmed to recognize all variants of murine DR3 protein in histological sections, we utilised the β-Gal gene expressed under the control of the murine DR3 promoter (Wang et al., 2001b), to investigate the regional and developmental pattern of putative DR3 protein expression in the brains of mice. DR3 gene expression was first detected neo-natally after d5 post-birth and was rapidly upregulated by d9 post-parturition (Fig. 1). This expression pattern was retained through early to late adulthood (Fig. 2A-2C). To explore the expression pattern of DR3 in adult DR3het and DR3ko mice, matched β-Gal and H&E stained coronal brain serial sections (10μm thickness) were compared allowing construction of DR3 promotor expression maps. Positionally, β-Gal expression was detected between the piriform cortex throughout the brain of transgenic mice to the caudal mesencephalon, disappearing at the transition to the cerebellum. In mice, the density and pattern of β-Gal staining was region specific: substantial numbers of β-Gal+ cells were present within the hippocampal and cortical regions, but expression was undetectable in the striatum, substantia nigra and cerebellum (Fig. 2A-2C). In the hippocampus, nearly all granular (dentate gyrus) and pyramidal cells (CA1-CA3 fields) stained densely for β-Gal whereas coloration between these layers was less frequent. In the cortex, β-Gal+ cells were diffuse but present in all layers (Fig. 2A and 2B). The β-Gal+ staining pattern found in the brains of DR3ko mice was largely mirrored in age-matched DR3het mice although weaker in intensity. Intriguingly, however, a several-fold greater number of β-Gal+ cells were found throughout the cortex of DR3ko compared to DR3het mice suggesting an attempt to express the DR3 gene in its absence (Fig. 2A).
To confirm the basal regional pattern of DR3 expression in the brain revealed by β-Gal, an adult DR3wt brain was micro-dissected into regions and DR3 mRNA expression assessed by RT-PCR. DR3 transcripts were expressed at high levels in the cortex, hippocampus and dentate gyrus but remained undetectable in the cerebellum of adult DR3wt mice consistent with DR3 localisation visualised by β-Gal expression studies (Fig. 2D and Supplemental Fig. 1). Some message was obtained from the DR3wt basal ganglia, but β-Gal staining was not observed in this region in DR3het and DR3ko mice. As expected, no expression of DR3 mRNA transcript was detected in DR3ko mice (Supplemental Fig. 1). We next used immunohistochemical studies to define the lineage of cells within the brain. In DR3het mice, analysis revealed that β-Gal expression co-localised almost exclusively with the neurone-specific marker, NeuN, and not with the astrocyte and glial cell marker, GFAP (Fig. 2E). These results indicate that DR3 expression is neuron-lineage specific, developmentally regulated, and regionally defined in the adult mouse brain.
We next sought to determine the level and pattern of TL1A mRNA expression, the only substantiated ligand for DR3 (Kaptein et al., 2000; Migone et al., 2002; Bossen et al., 2006), in the brains of adult mice. Interestingly, expression of transcripts for TL1A mirrored that of DR3 with the highest expression found in the cortex, and the lowest expression detected in the cerebellum (Fig. 2D). More moderate TL1A mRNA expression was found in the hippocampus, colliculi, basal ganglia, and dentate gyrus (Fig. 2D). The survival, structure, and function of neurons in the brain is known to be critically dependent on glial cells (Fiacco et al., 2008; Nave and Trapp, 2008). Several studies have identified astrocytes as being responsible for intensive production of several TNF family ligands following a range of stimuli in vitro and in vivo (Sawada et al., 1989; Badie et al., 2001; Shin et al., 2002; Yan et al., 2007). We therefore purified astrocytes from the cortex of DR3wt mice and examined whether TL1A mRNA was expressed by these cells ex vivo. Cultured astrocytes expressed low levels of transcript for murine TL1A, which was upregulated rapidly by TNFα, suggesting that these cells may be responsible for constitutive low level provision of TL1A to local neurons within the cortex (Supplemental Fig. 2). We also examined whether the expression levels of a range of other TNFRSF members (TNFR1, FAS, DR4/6, DR5 and p75NTF) or the DR3 modulator, silencer of death domain (SODD) might be altered in the absence of DR3 expression. Expression of mRNA for these proteins was comparable between DR3het and DR3ko mice by RT-PCR, indicating that loss of neuronal DR3 expression is not compensated for by up-regulation of other death domain TNFRSF members (Supplemental Fig. 3). Collectively, these studies suggest that TL1A is expressed by glial cell populations in the absence of inflammation, while expression levels of other TNF family members in the cortex of DR3ko mice remain unaltered. The presence of constitutive cortical TL1A expression in the absence of inflammation, suggests that TL1A/DR3 signalling may play an important role in normal neuronal function or maintenance in the cortex.
To assess the role of constitutive DR3 expression in normal brain function, the behaviour of DR3wt, DR3het and DR3ko mice was monitored and assessed during aging. Initial observations revealed that DR3ko mice developed a progressive behavioural disorder characterised by abnormal gait, rapid head movement, apparent disorientation, dyskinesia and body tremor that was triggered by environmental change such as handling, none of which were seen in DR3wt or DR3het mice. Occasionally, early aged founder mice were also found to suffer from seizures. Behavioural abnormalities were detectable in a proportion of DR3ko mice as young as 4 months and the sex-independent disorder was 100% penetrant by 18 months of age (Fig. 3). To quantify this defect, mice were timed traversing a perspex corridor and measurements of stride length, width and distance between hind and forepaw placement (overlap) were made for mice aged 3 (young) and 24 months (old). While run times between young DR3het, DR3ko, and old DR3het mice did not differ, old DR3ko mice took significantly longer to complete their runs (DR3ko 6.8 ± 1.3 sec vs DR3wt 3.4 ± 0.7 sec; F(1,10) = 5.84, p<0.04) (Fig. 4A). An age-dependent difference was also detected in the gait measurements of DR3ko mice, a representation of which is shown in Figure 4B. Only old DR3ko mice exhibited significantly shortened forepaw stride lengths (DR3ko 5.1 ± 1.1 cm vs DR3wt 6.3 ± 0.5 cm; F(1,10) = 6.17, p<0.04), broader hindpaw stride width (DR3ko 3.0 ± 0.3 cm vs DR3wt 2.6 ± 0.2 cm; F(1,10) = 9.84, p<0.02) and decreased overlap relative to matched controls (DR3ko 1.4 ± 0.6 cm vs DR3wt 0.7 ± 0.2 cm; F(1,10) = 8.11, p<0.02) (Fig. 4C). We also explored whether the balance of DR3ko mice might also be impaired by defective motor control as found for some models of neurological disease (Fernagut et al., 2004; Truong et al., 2006). Aged DR3het mice all traversed the 60 cm long balance beam on all three runs in an average time of 11.4 ± 0.6 sec. By contrast the DR3ko mice had much greater difficulty completing the task: only two of the 8 mice succeeded to cross the beam within the 120 sec observation period on all 3 runs, 3 failed on 1 or 2 occasions to cross, and 3 failed on all three occasions (Fig. 5A). For purposes of analysis, failed runs were allocated a maximum crossing time of 120 sec, yielding a group mean latency to cross of 83.0 ± 15.7 sec, which was significantly longer than that taken by the DR3het group (F(1,13) = 18.09, p<0.003) (Fig. 5B). Whilst loss of footing by well-balanced adult DR3het mice was rare, movement by DR3ko mice was marked by frequent imbalance, foot slippage and stationary behaviour (Fig. 5C).
We next examined the movement and exploratory behaviour of DR3ko mice by comparing the activity of transgenic mice during open field tests using a graduated 1m perspex square. Compared to 24-month DR3het controls, similarly aged DR3ko mice exhibited hyperactivity and increased complexity of movement characterised by rapid, abrupt changes in forward direction and path-crossing with some animals showing strong circling behaviour (Supplemental Video 1-3; Fig. 6A). In a 10 min observation period, DR3ko mice traversed an average of 250% more accumulative open space, covering 89 ± 17 m relative to DR3het controls, which traversed 35 ± 5 m (F(1,10) = 8.87, p<0.02) (Fig. 6B). The relative complexity of DR3ko mice open field activity also increased compared to DR3het mice as determined by the frequency mice crossed their own path, with DR3ko mice averaging this readout 286 ± 86 times over 10 mins, ~14 times more frequently than their DR3het counterparts who averaged this 20 ± 6 times (F(1,10) = 9.47, p<0.02) (Fig. 6B). This increased locomotor complexity was not due to their hyperactivity as the average distance travelled before a crossover event was significantly shorter in DR3ko mice compared to DR3het controls (DR3ko 0.41 ± 0.07 m vs DR3wt 2.5 ± 0.5 m; F(1,10) = 16.83, p<0.003) (Fig. 6C). No directional preference was observed in the circling behaviour of individual mice during open field tests irrespective of age or sex. Similar results were obtained with DR3ko mice that had been crossed with CD1 (outbred) mice for 2 generations (data not shown), demonstrating that the dysfunction was not due to a specific genetic background. Collectively, these results confirm that DR3ko mice develop significant motor coordination problems, hyperactivity and abnormal exploratory behaviour in an age-dependent fashion.
We next determined whether the behavioural abnormalities exhibited by aged DR3ko mice were associated with altered levels of neurotransmitters in the brain. Brains from 3-month and 18-month old mice were micro-dissected into the cortex, striatum, hippocampus and cerebellum, and the levels of neurotransmitters assessed by high performance liquid chromatography (HPLC). Brains from DR3ko mice appeared histologically normal being devoid of apparent cellular infiltrates, lesions/plaques, upregulated inflammatory markers and apparent inflammation as assessed by immunohistochemistry (unpublished observations). Weights of whole and individually dissected regions of brain were also similar between DR3ko and DR3het animals (Supplemental Fig. 4). Young DR3ko and DR3wt mice of 3 months age showed no significant differences in neurotransmitter levels in the brain, but strikingly, we found that dopamine levels within the striatum of aged 18-month DR3ko mice were 60% higher than their DR3het counterparts (DR3ko 15546 ± 1163 ng/g tissue versus DR3het 9768 ± 2383 ng/g tissue; F(1,9) = 5.32, p<0.05), while serotonin was 4-fold lower (DR3ko 77 ± 21 ng/g tissue versus DR3het 328 ± 81 ng/g tissue; F(1,9) = 10.63, p<0.01) (Table 1). These changes could not be explained by a failure of dopamine breakdown as the ratios of dopamine to dopamine metabolites (DOPAC, HVA) were unchanged (Table 1 and Supplemental Fig. 5). Furthermore, it could not be explained by upregulation of tyrosine hydroxylase (TH), the primary enzyme responsible for dopamine production, as TH levels throughout the brain were comparable between DR3ko and DR3het controls as assessed by immunohistochemistry on coronally matched sections (Fig. 7A, 7B). These data demonstrate that despite an absence of overt neurological pathology, aged DR3ko mice have profound differences in striatal neurochemistry.
Current literature suggests that DR3 can control cell fate by initiating either proliferative or apoptotic signalling (Chinnaiyan et al., 1996; Kitson et al., 1996; Marsters et al., 1996; Migone et al., 2002). We therefore examined whether neuronal lifespan might be altered in the absence of DR3 expression in vivo by examining neuron numbers in aged mice. The number of NeuN+ cells present in aligned sections from the brains of aged DR3het and DR3ko mice showed no significant difference following 2-D histological topographic analysis (Fig. 8A). The area and thickness of individual layers or regions of the brain as calculated by computer topography was also found to be similar between DR3het mice and their controls (Supplemental Table 2). No difference was also found in the number of apoptotic neurons in the brains of adult DR3ko mice, compared to controls, which were barely detectable (unpublished observations) in agreement with others (Yang et al., 2008). Together, these data suggest that DR3 plays no overt role in the normal determination of neuron lifespan in adult mice in vivo.
Given that aged DR3ko mice had normal neuron numbers and DR3 was not expressed in the striatum, we next hypothesised that corticostriatal innervation might be altered by the loss of neuronal DR3 expression as has been recently observed for another TNFRSF member, GITR (O'Keeffe et al., 2008). This was directly assessed by injection of the retrograde axonal tracer, fluorogold (FG), into the striatum of mice. The cortex of 18-month old DR3ko mice had significantly lower numbers of FG labelled pyramidal cells compared to similarly treated control animals (Fig. 8B and 8C). Overall, more than 25% of corticostriatal contact had been lost in aging DR3 deficient mice in the absence of continued cortical DR3 expression by neurons as indicated by retrograde tracing (DR3ko 74.7 ± 2.7 % versus DR3wt 100 ± 8.5 % of normal cortical density; F(1,6) = 8.25, p<0.03). No significant differences were observed between young 3-month old DR3wt (100 ± 8 %) and DR3ko (95 ± 2 %) mice (Fig. 8C). These results demonstrate that continued neuronal DR3 expression is important for the normal, long-term maintenance of neuronal innervation between the cortex and striatum in adult mice in the absence of appreciable neuron loss. It is less likely that there is a developmental deficit since there was no differences in innervation density between 3-month animals.
In this study, we demonstrate for the first time that DR3 is critically required for maintaining neural connectivity between the cortex and striatum during aging, which is necessary for normal motor control function. DR3ko mice develop a progressive locomotor disorder defined by deficits in coordinated movement and gait, which emerges during early adulthood, appears sex-independent and is 100% penetrant. Importantly, advancing loss of motor control in DR3ko mice occurred through a relatively passive process without the induction of apparent inflammation, alteration to other TNFRSF brain expression levels or radical changes in neuron number. Thus, we propose that low endogenous TL1A stimulation of DR3+ cortical neurons stimulates the maintenance of inter-neuronal contact with adjoining dopaminergic or serotonergic striatal neurons. Supporting this, we observed in vitro expression of TL1A mRNA from cultured astrocytes isolated from the cortex, the primary site of TL1A transcript expression in vivo. How might TL1A promote DR3 dependent innervation? One possibility is that TL1A may contribute directly to axon growth or dendrite formation in aging neurons such as occurs following FasL treatment of embryonic neurons (Zuliani et al., 2006) and GITRL treatment of neonatal sympathetic neurons (O'Keeffe et al., 2008). Alternatively, TL1A may increase neuron excitability or alter ion channel signalling as has been shown for TNFα (Diem et al., 2001; Ogoshi et al., 2005; Ozaktay et al., 2006; Czeschik et al., 2008). In such cases, more frequent neuronal stimulation would be predicted to drive new synapse/dendrite formation and stabilization (De Roo et al., 2008). Importantly, our results suggests that the maintenance of adult neuronal corticostriatal projections formed earlier during development and critical for motor control is dependent on the continued reception of low level DR3 signalling by neurons involved in these contacts.
The expression of transcripts for TL1A throughout many parts of the brain in a region specific manner is also a novel finding. Prior to this study, TL1A has been reported to be found in serum (Bamias et al., 2008), HUVECs (Yang et al., 2004), myeloid and T cells (Bamias et al., 2003; Cassatella et al., 2007; Prehn et al., 2007; Meylan et al., 2008). TL1A has been reported to have roles in mucosal immunity (Papadakis et al., 2005), inflammatory bowel disease (Bamias et al., 2006; Takedatsu et al., 2008), inflammatory arthritis (Borysenko et al., 2006; Cassatella et al., 2007; Bull et al., 2008), renal inflammation (Al-Lamki et al., 2008), asthma (Fang et al., 2008; Meylan et al., 2008) and autoimmune encephalitis (Meylan et al., 2008; Pappu et al., 2008). Since the brains of DR3 deficient animals have normal cell numbers and appear devoid of any inflammation or apparent neuropathology, this suggests that cerebral mechanisms exist to dampen neuronal DR3 responsiveness, which is likely to be very threshold or age sensitive. This might be expected as TL1A seems to be constitutively produced by cells within the brain in regions where DR3+ neurons cohabit.
Progressive behavioural defects in DR3ko mice paralleled the differential expression of striatal neurotransmitters with increased dopamine and a corresponding serotonin reduction. Dopamine signalling appears essential for motivated behavior as shown by severe defects in feeding, motor control and reward-based learning apparent in Dopamineko mice (Zhou and Palmiter, 1995; Palmiter, 2008). Serotonin receptor family deficient mice also display altered locomotion and behavior (Lesch et al., 2003). Moreover, serotonin and dopamine imbalances are prominently associated with human neurological disorders ranging from Parkinson's disease to schizophrenia and circling in mice (Nieoullon and Coquerel, 2003; Ishiguro et al., 2007; Di Matteo et al., 2008). The biosynthesis of dopamine occurs in a two stage process in which L-tyrosine is first converted to L-DOPA, by the enzyme tyrosine hydroxylase (TH), and then L-DOPA is converted to dopamine, by the enzyme dopa decarboxylase (Zhou and Palmiter, 1995). Striatal dopamine levels appear dependent on production by DA neurons projected from the substantia nigra (SN) as changes in striatal dopamine often reflect alteration to SN neuron number (Kramer et al., 2007). In aged DR3ko mice, however, we show that striatal dopamine levels were elevated to 160% in the absence of any change to brain neuron numbers or alteration in the level or pattern of TH expression. We also found that the levels of two products generated from the breakdown of dopamine, DOPAC and HVA were found in normal ratios suggesting that a failure of striatal dopamine metabolism was not responsible for increase levels of this neurotransmitter. It has been suggested that continued dopamine expression is important for the correct maintenance of corticostriatal synapses as unilateral destruction of dopaminergic striatal input leads to innervation failure (Arbuthnott et al., 2000). However, whether a specific loss of corticostriatal innervation can induce a corresponding upregulation of striatal dopamine remains unclear. Alternatively, neurotransmitter dysregulation in DR3ko mice may reflect the absence of direct DR3-dependent alteration of neurotransmitter production or subsequent synaptic sensitivity as triggered by TNFα (Schafers and Sorkin, 2008). Both models are consistent with the hypothesis that behavioral defects in dopamine-dependent disorders may reflect corticostriatal pathway dysfunction, as found in DR3ko mice, rather than just changes to cortical activity (Costa et al., 2006).
Striatal serotonin levels were also reduced by 73% in adult DR3ko mice. There is evidence to suggest that excessive dopamine production can inhibit serotonin production by serotonergic neurons in the striatum in vivo leading to behavioral defects. Many Parkinson's disease patients receiving long term L-DOPA treatment suffer motor control loss associated with dopamine dysregulation (Cenci and Lindgren, 2007). Similarly, dopamine uptake, production, storage, and release can supplant and deplete serotonin in striatal serotonin neurons following chronic L-DOPA treatment in several models (Kannari et al., 2006; Yamada et al., 2007; Carta et al., 2008). We therefore suggest that excessive striatal dopamine production in DR3ko mice is down regulating serotonin expression and that neuronal DR3 signaling is likely to be a major inhibitory dampener suppressing dopamine induction in vivo.
The role of basal TNFRSF member brain expression and function is an emerging field with few published studies. In vitro, select TNFSRF members have been shown to promote nerve growth and function (Shao et al., 2005; Czeschik et al., 2008; Hayashi et al., 2008; Hou et al., 2008; O'Keeffe et al., 2008). Constitutive brain expression of some TNFRSF receptors has also been reported (Yan and Johnson, 1988; Pan et al., 1998; Eby et al., 2000; Kojima et al., 2000; Tan et al., 2002; Pispa et al., 2003; Shao et al., 2005; Zuliani et al., 2006; Hamill et al., 2007; Catts et al., 2008; Harry et al., 2008; Hou et al., 2008; O'Keeffe et al., 2008), but the neurological function of most members remains unknown. The extensive behavioral and neurochemical defects exhibited by DR3ko mice are unique amongst TNFRSFko mice. For example, TNFR1ko mice have normal striatal dopamine/metabolite levels (Leng et al., 2005) and locomotor activity, exhibiting standard behavioral responses in several tests (e.g. open field and Porsholt swim) (Simen et al., 2006; Quintana et al., 2007; Baracchi and Opp, 2008). Why DR3ko mice, amongst TNFRSF deficient animals, uniquely develop severe and progressive neurological defects requires further study but probably reflects receptor differences in function and endogenous age related expression. In this respect, we firstly show that continued neuronal DR3 signalling functions by uniquely promoting region-specific (cortico-striatal) brain innervation. Secondly and regardless of the mechanism, basal neuronal DR3 appears to functionally regulate the expression of two key neurotransmitters required for movement and behavior, dopamine and serotonin. Thirdly, we note that stable postnatal neuronal DR3 expression appears more intense and widespread in the cortex and hippocampus relative to most TNFRSF members in brain regions with motor control pathway function. No other TNFRSF member has been similarly reported to functionally promote the stabilization of pre-existing adult neural pathways and regulate striatal neurotransmitter expression under normal conditions in vivo. TNFα though has been shown to act as a gliotransmitter, altering synaptic transmission (Stellwagen et al., 2005), controlling synaptic strength (Beattie et al., 2002) and scaling (Stellwagen and Malenka, 2006), while other pro-inflammatory cytokines such as IL-1 can influence synaptic plasticity (Ross et al., 2003). The interactions between TL1A and these cytokines in the brain are poorly understood and remain an important area of further investigation. Our data is consistent with the suggestion that the key neurological function of individual TNFRSF receptors is likely to be developmentally, functionally and regionally compartmentalized within the brain.
In this report, we identify neuronal DR3 signaling as being mandatory for normal striatal neurotransmitter expression and maintenance of corticostriatal neural pathways, which are essential for normal motor control function in the aging adult. How DR3 signalling provides pathway stabilisation cues to these neural networks remains an important issue to be resolved, however the observation that aging DR3ko mice acquire defective neurotransmitter expression and progressive motor control loss provides a unique insight into this pro-inflammatory receptor. Importantly, this study demonstrates that DR3 plays a key non-redundant role in behavior and normal adult brain homeostasis, further expanding upon the essential roles that TNFSFR members play within the mammalian body. As such, we identify DR3 as a primary molecular target for future neuromodulatory and neurodegenerative disease research.
This work was supported by Medical Research Council Career Establishment (G0300180) and Collaboration (G0500617) Grants awarded to ECYW. MIR was supported by a BBSRC CASE studentship (C/05060) in collaboration with Glaxo Smith Kline (GSK). AMT was supported by a Wellcome Trust programme grant (064232). We thank Peter Maycox and Isabel Benzel at GSK for helpful discussion and support.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.