Down Syndrome (DS) is caused by trisomy of
A large number of genes are simultaneously expressed at abnormal levels in DS, therefore, it is a challenge to determine which genes contribute to specific abnormalities, and then identify the key molecular pathways involved. We are advocates of the approach articulated by Nadel  - that a careful and detailed analysis of the clinical defects in humans be followed by the creation of mouse models that over-express only some of the genes triplicated on
The focus of this chapter will be to provide insight into
2. The RCAN gene family
3. General tissue and cellular expression of RCAN1
RCAN1-1 and RCAN1-4, the most predominantly expressed isoforms, are under the control of different promoters and are therefore likely to have different regulatory mechanisms and possibly even different functions. For example,
The subcellular location of RCAN1 protein was initially determined using tranfection of a RCAN1-GFP protein construct in C2C12 cells, a mouse myoblast cell line. RCAN1 protein was located in both the nuclear and cytosolic compartments and in the absence of treatments to activate the calcineurin signalling pathway, resided predominantly in the nucleus . Various physiological and biochemical stresses have been demonstrated to influence the location of RCAN1 within a cell. For example, under normal circumstances RCAN1 was located within the nuclear compartment in various cell lines, including HT-1080 fibrosarcoma and I251 astroglioma cells. However, when these cells were subjected to oxidative stress, RCAN1 protein was redistributed to the cytoplasm . The same observation was made following activation of the calcineurin signalling pathway, which resulted in the translocation of RCAN1 from the nucleus into the cytosolic compartment .
4. Functional domains of the RCAN1 protein
Initial studies found that both RCAN1 isoforms encode a proline rich protein consisting of a putative acidic domain, a serine proline motif, a putative DNA binding domain and a proline rich region typical of a SH3 domain ligand [22, 28]. These structural motifs are typically seen in proteins involved in transcriptional regulation and signal transduction. A more recent study on RCAN1 proteins in dozens of species revealed 4 highly conserved regions separated by other regions that are less well conserved. These four regions consist of: a region at the amino terminus capable of forming an RNA recognition motif; the gene family signature domain consisting of the highly conserved SP motif; a PxIxIT-like domain (x represents any amino acid) and a C-terminal TxxP motif  (see Figure 1). The functions of these highly conserved regions in RCAN1 proteins are yet to be fully explored.
The most highly conserved region in the RCAN1 protein is the SP motif. This motif is similar to that present in NFAT proteins .
Site-directed mutagenesis studies have shown that phosphorylation of the RCAN1 protein regulates its function, subcellular location and stability. Indeed, RCAN1 can be phosphorylated by various kinases at a number of different sites to change its activity towards calcineurin. For example, the serine residue within the SP domain at position 112 (Ser112) (Ser167 in RCAN1-1) is variously phosphorylated by BMK1 , NIK  and DYRK1  and acts as a priming site for subsequent phosphorylation at Ser108 (Ser163 in RCAN1-1) by GSK-3   . Phosphorylation by TAK1 at Ser94 and Ser136  and by DYRK1A at Thr193  also change the activity of RCAN1 towards calcineurin (see later). NIK-mediated phosphorylation  or phosphorylation by PKA  augmented the half-life of RCAN1 protein. And, phosphorylation of a threonine residue (Thr166 in RCAN1-4) in the SH2 domain controlled its subcellular localisation since exchanging the threonine for an alanine resulted in an accumulation of RCAN1 protein within the cytoplasm . Thus, nuclear localisation of RCAN1 is controlled, at least in part, by phosphorylation.
Other studies have shown that RCAN1 is cleaved by calpain and this cleavage appears to increase the stability of the protein by decreasing its proteasome-dependent degradation . Further, the cleavage of RCAN1 by calpain also affects its interactions with other proteins. For example, cleavage of RCAN1-4 by calpain abolished its ability to bind to Raf-1 . Yet another pathway involved in the post translational regulation of RCAN1 is the ubiquitin-proteasome system (UPS). The UPS is important in the regulation of protein turnover in response to changing cellular conditions and facilitates the degradation of defective proteins . Ubiquitin is a polypeptide able to bind to lysine residues on proteins targeted for degradation. This binding occurs through sequential steps mediated by ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin-protein ligase (E3) . Following this sequence of events, the 26s proteasome is able to recognise and degrade the poly-ubiquinated protein. The first evidence to suggest that RCAN1 was degraded by the ubiquitin pathway came from yeast two hybrid and co-immunoprecipitation experiments which found that RCAN1-4 interacted with ubiquitin . More recent studies demonstrated that RCAN1 interacts with other members of the UPS, including, Skp1, Cullin/Cdc53, F-box protein Cdc4 (SCFCdc4)  and SCFβ TrCP1/2 . The interaction between RCAN1 and the UPS is not only important in regulating turnover of the RCAN1 protein but may also influence its function. For example, increased degradation of RCAN1 by SCFCdc4 diminished its ability to inhibit calcineurin signalling .
5. RCAN1 function—Signal transduction pathways
5.1. The calcineurin pathway
The calcineurin pathway plays an integral role in the development and homeostatic regulation of a number of different cell types, including immune cells and neurones. The pathway is activated by increases in intracellular calcium (Ca2+) due to oxidative stresses, chemical-mediated calcium increases and in response to biomechanical strain . An increase in intracellular Ca2+ leads to the activation of calmodulin, which forms a complex with calcineurin to activate its phosphatase function. Activated calcineurin then dephosphorylates cytosolic NFAT leading to its translocation to the nucleus where it complexes with GATA-4  allowing DNA binding and facilitation of the transcription of numerous gene targets .
RCAN1 interacts directly with calcineurin  . Calcineurin is a heterodimer, consisting of a catalytic A subunit and a calcium binding regulatory B subunit . RCAN1 is able to bind to the A subunit in a linker region between the calcineurin A catalytic domain and the calcineurin B binding region . Deletion of the carboxyl-terminal half of the catalytic domain of calcineurin A abolished binding with RCAN1, indicating that this region was critical for the interaction . Studies with RCAN1 have shown that exon 7 is able to bind to and regulate the activity of calcineurin and this binding occurs with a very high affinity . While binding of RCAN1 to calcineurin did not interfere with the interaction between calcineurin and calmodulin, it is believed to interfere with the ability of calcineurin to bind NFAT by competing with the NFAT binding site . Indeed, when
Interestingly, activation of calcineurin signalling induces
As more and more studies have emerged on RCAN1 and the propagation of calcium signals in the cell, it has become clear that the role of RCAN1 is not always to inhibit the calcineurin pathway. While the earliest studies found RCAN1 to negatively regulate the pathway, in other circumstances it seems to facilitate calcineurin activity. Indeed, contrary to expectations it was found that the absence of
These apparently paradoxical actions of RCAN1 may be explained, at least in part, by its cellular concentration, its nuclear or cytosolic localisation and/or its phosphorylation status     . For example, the abundance of RCAN1 in the cell may determine its ability to either enhance or inhibit calcineurin signalling. Low or intermediate levels of RCAN1 were shown to facilitate calcineurin signalling while very high levels of over-expression were inhibitory, suggesting that RCAN1 oscillates between stimulatory and inhibitory forms depending on its concentration  . In contrast, in another study, the functional role of RCAN1 was found to change in a dose-dependent fashion, but in the opposite direction to the aforementioned studies – RCAN1 was an inhibitor at low levels but a facilitator when levels were high . Another study indicated that 4 highly conserved domains in the RCAN1 protein were important in determining its activity towards calcineurin. Specifically, that preferential binding of RCAN1 to calcineurin prevented NFAT binding resulting in inhibition of calcineurin signal transduction due to competition between RCAN1 and NFAT for calcineurin docking sites . This preferential binding occurred in the presence of high levels of Rcan1 and required the LxxP domain within the SP motif and the PxIxIT domain . Conversely, when Rcan1 was expressed at lower levels, the protein was able to stimulate calcineurin signalling. This stimulatory effect required the LxxP and ExxP domains within the SP motif as mutations within both of these domains prevented stimulation.
Other studies have suggested that it is the phosphorylation status of RCAN1 that determines its action as either an inhibitor or facilitator of calcineurin activity. A study in yeast found that for Rcan1 to facilitate calcineurin signalling it required phosphorylation of both serine residues located within the SP motif by a priming kinase (in this case MAPK) and Mck1, a member of the glycogen synthase kinase 3 (GSK-3) protein family. When the serines were mutated to alanines or in the absence of Mck1, Rcan1 was no longer able to stimulate calcineurin signalling resulting in inhibition . Phosphorylation by TAK1, DYRK1A and NIK all switch RCAN1 from an inhibitor to a calcineurin facilitator   . At odds with most studies, phosphorylation of the serine residues within the SP motif of RCAN1 was reported to enhance its ability to inhibit calcineurin .
In summary, although the mechanisms responsible for the dual role of RCAN1 in the calcineurin signalling pathway are still under investigation, the results so far indicate that the primary function of RCAN1 is to facilitate calcineurin activity and this occurs when RCAN1 is expressed at lower or physiological levels. On the other hand, when RCAN1 is highly expressed, it has a secondary role of inhibiting calcineurin signalling by interfering with the interaction between calcineurin and NFAT.
5.2. GSK–3 signalling
Numerous studies outlined above have shown that GSK-3 phosphorylates RCAN1 to regulate its function. Interestingly, GSK-3 activity can also be regulated by RCAN1. PC-12 cells over-expressing RCAN1 displayed an increase in the absolute levels of GSK-3β protein, which in turn increased its kinase activity towards Tau . Tau protein is a known target of GSK-3 which in its hyperphosphorylated form has been implicated in the aetiology of Alzheimer’s disease . Exactly how RCAN1 regulates the abundance of GSK-3 remains undetermined, but it seems that RCAN1 is acting at a post-transcriptional level as the amount of
5.3. The MAPK/ERK signalling pathway
The MAPK/ERK signalling pathway mediates signal transduction from cell surface receptors to downstream transcription factors. This pathway plays a role in a number of cellular processes including proliferation, growth, motility, survival and apoptosis . As indicated above, MAPK was able to phosphorylate RCAN1 at S112 within the SP motif to prime its subsequent phosphorylation by GSK-3. Moreover, the same study demonstrated that phosphorylation of RCAN1 by MAPK allowed RCAN1 to become a substrate for calcineurin , thus introducing a further level of control to keep the pathway operating at an optimal level.
5.4. The NFκβ inflammatory pathway
RCAN1 is also able to regulate the Nuclear factor κβ (NFκB) signalling pathway. NFκB is a transcription factor that regulates target genes involved in many physiological processes, including immunity, inflammation, cancer, synaptic plasticity and memory. Under normal circumstances, NFκB exists as a dimer and is sequestered in the cytoplasm through its interaction with an inhibitory molecule known as Inhibitor of κB (IκB). Upon stimulation of the NFκB signalling pathway, IκB is degraded by the ubiquitin/proteasome pathway releasing its inhibitory action on NFκB . Degradation of IκB allows NFκB to translocate to the nucleus where it acts to induce the expression of various target genes including the inflammatory genes cyclooxygenase-2 (
Studies have also linked RCAN1 to NFκB signalling via other members of the pathway. For example, RCAN1 is able to negatively regulate the mRNA expression of NFκB inducing kinase (
Angiogenesis is a physiological process involving the growth of new blood vessels essential for embryonic development as well as growth and development throughout life. This process has also been associated with disease states including inflammation, tumourigenesis and cardiovascular disease . Angiogenesis is orchestrated by a balance between pro-angiogenic factors and angiogenic inhibitors . A critical mediator of angiogenesis is Vascular endothelial growth factor (VEGF) which acts to stimulate angiogenesis and vascular permeability [73-75]. VEGF stimulation of cells causes the rapid activation and translocation of NFAT into the nucleus which in turn results in the up regulation of numerous genes associated with angiogenesis . A number of studies have implicated RCAN1 in angiogenesis. Early studies found that
Both major RCAN1 isoforms are involved in angiogenesis and appear to be regulated by different mechanisms. When human endothelial cells were treated with VEGF, there was an induction of
A number of reports have suggested that RCAN1-1 and RCAN1-4 may play opposing roles in angiogenesis, where RCAN1-1 appears to be pro-angiogenic and is capable of inducing the formation of new blood vessels, while RCAN1-4 inhibits angiogenesis and vessel formation. For example, siRNA-mediated silencing of
6. The consequences of RCAN1 over-expression in the DS brain
6.1. Down syndrome and the neural system
DS is the leading genetic cause of intellectual impairment in the general population and is thought to contribute to around 30% of all cases of moderate to severe mental retardation . Mental retardation in DS is characterised by behavioural and cognitive impairments which include low IQ, language deficits and defects in both short and long term memory. Later these deficits are compounded by the early onset of dementia .
People with DS exhibit a reduced performance on a number of different tests designed to demonstrate short term or working memory, including visual perception, visual imagery and spatial imagery tasks . Long term memory is also affected by DS with both implicit (defined as improvement in perceptual, cognitive or motor tasks without any conscious reference to previous experience) and explicit (intentional recall or recognition of experiences or information) memory impaired . In addition to the cognitive defects observed throughout life, neuropsychological tests showed that there is a cognitive decline in DS individuals with age and these cognitive changes equate to those observed following the onset of dementia . DS participants with early stage dementia displayed severely diminished long term memory as well as a decreased ability to retrieve stored information compared with the non-demented DS controls . The decline in these forms of cognition, particularly the ability to form new long term memories, is analogous to the cognitive deterioration seen in early to moderate Alzheimer’s disease (AD) . Interestingly, the cognitive defects that characterise DS are associated with hippocampal-based learning and memory while prefrontal-mediated executive function and cognition remain relatively unaffected .
The cognitive impairments in DS are accompanied by many neuro-morphological changes. Individuals with DS have a significant reduction in brain weight and volume , despite brain weight falling within the normal range at birth . DS brains have a shorter anterior-posterior diameter, a reduction in the size of the frontal lobes, a flatter occipital lobe and a smaller brain stem and cerebellum . The anterior and posterior corpus callosum regions and hippocampus are also smaller [92-95]. The hippocampus is a key brain structure involved in learning and memory and many of the behavioural and cognitive defects seen in DS are hippocampal-dependent . The difference in hippocampal volume is most likely due to various structural abnormalities, including a decrease in the mean area of the dentate gyrus (DG) and inadequate migration of cells into the pyramidal cell layer . Notably, in adults there is an additional age-related decrease in the volume of the hippocampus, most likely due to some degree of neurodegeneration .
Smaller brains in DS individuals probably result from a reduction in the total number of neurones, with certain regions preferentially affected. DS brains exhibit a decrease in neuronal density by adulthood of between 10-50% . The cortex of DS adults exhibits decreases in neuronal number and density in addition to abnormal distribution of neurones . This same pattern of neuronal loss was also observed in the hippocampus and visual cortex. Interestingly, DS foetuses exhibited the same pattern of neuronal development as normal foetuses, with similar neuronal morphology, dendritic spine number and density . However shortly after birth defects were evident and became more pronounced with age . This indicates that something happens after birth which results in alterations in neuronal number and morphology. Using Golgi staining which allows for the visualisation of neurones including their cell bodies, axons, dendrites and spines, the brains of DS infants exhibited shorter basilar dendrites with a significant decrease in the absolute number of spines , which was postulated to correlate with a 20-35% decrease in surface area per synaptic contact . Why and how this decline in neuronal development occurs is currently undetermined. These same defects were observed in adults with DS, who exhibited decreased dendritic branching, dendrite length and spine density . Biochemical examination of adult DS brains also revealed a significant reduction in the concentrations of various neurotransmitter markers including, noradrenaline, serotonin or 5-hydroxytraptamine (5-HT) and choline acetyltransferase (ChAT) [102, 103], again signifying neuro-functional deficits in the brain.
On top of the neurodevelopmental problems associated with DS, all individuals with the disorder develop the neuropathological and neurochemical changes associated with AD by the third decade of life . This includes the accumulation of amyloid β (Aβ), formation of hyperphosphorylated Tau-containing neurofibrillary tangles (NFT) and senile plaques. The progression of AD-neuropathology is analogous in both DS and AD, despite occurring decades earlier in DS .
6.2. RCAN1 in the brain
RCAN1 has been implicated in development and function of the brain.
Western blot analysis using an antibody designed to detect both RCAN1-1 and RCAN1-4 proteins found that the two isoforms were differentially expressed in the adult mouse brain. RCAN1-1 was abundant throughout the brain, with the highest levels of expression detected in the cortex and hippocampus [20, 54, 106]. RCAN1-4 was generally found at lower levels in the hippocampus, striatum, cortex and prefrontal cortex . Similar results have been observed in the adult human brain where RCAN1-1 was most highly expressed in the cerebral cortex, hippocampus, substantia nigra, thalamus and medulla oblongata . It is worth noting that while one study indicated that both isoforms of RCAN1 were located exclusively within neurones and not in astrocytes or microglial cells , another study found a wider distribution pattern , with RCAN1-1 and RCAN1-4 detected in multiple cell types including astrocytes and microglia. The highest levels of expression were observed in neurones . Moreover, RCAN1-1 was also detected in primary glial-like cell cultures containing microglial cells and expression of RCAN1-4 was strongly induced following calcium stress .
Experimental evidence suggests that RCAN1 has a role in brain function. For example, studies on the RCAN1 orthologue in
Similar behavioural abnormalities were observed in RCAN1-KO mice. While the absence of
These behavioural deficits in RCAN1-KO mice were accompanied by abnormal synaptic transmissions and impaired long term potentiation (LTP). LTP is a form of synaptic plasticity hypothesised to be a biological substrate for some forms of memory . Two forms of LTP can be examined: early-component LTP (E-LTP), a weak and short-lived enhancement of synaptic transmission; and late-component LTP (L-LTP) which is a robust enhancement of synaptic transmission lasting many hours [110, 111]. Paired-pulse facilitation (PPF) is also a component of LTP and is a measure of pre-synaptic short-term plasticity and neurotransmitter release . Absence of RCAN1 did not affect the basal level of synaptic transmission but did result in a reduction in PPF compared with the WT controls, suggesting that pre-synaptic short term plasticity was affected by the lack of
The strongest evidence to suggest a role for RCAN1 in the neurological defects observed in DS comes from a recent study by our group examining
RCAN1 has also been shown by our group to be involved in neurotransmission. Using chromaffin cells cultured from the adrenal gland as a model for the neuronal system, cells from both RCAN1-TG and RCAN1-KO mice displayed a reduction in neurotransmitter release. Our study demonstrated that the normal function of RCAN1 was to regulate the number of synaptic vesicles fusing with the plasma membrane and undergoing exocytosis, and the speed at which the vesicle pore opens and closes . Although our study showed that the final outcome was the same whether RCAN1 was in excess or deficit, increased expression of
6.3. RCAN1 in neurodegeneration
Although it has not been proven, there is circumstantial evidence to suggest that RCAN1 plays a role in neurodegenerative conditions (other than DS). For example, Northern blot analysis of human brain samples found that
While these observations are intriguing, the question remains, what effect does increased RCAN1 expression have on the ageing brain and does it play a role in AD-like neuropathology? While this question remains unanswered, there are a number of possible reasons as to why increased RCAN1 expression might lead to neurodegeneration. One proposed explanation invokes a possible relationship between elevated RCAN1 expression, AD-like neurodegeneration and Tau protein. Tau is involved in the stabilisation of the microtubule networks within neurones and its hyperphosphorylation has been linked to the pathogenesis of AD. Tau can be phosphorylated by a number of different kinases, including GSK-3β and Ca2+/calmodulin-dependent protein kinases (CaMK). Hyperphosphorylation of Tau is detrimental and can lead to AD neuropathology, including formation of NFT [114-116]. During normal cellular processes, there is a proteasome-dependent degradation of Tau protein but when Tau becomes hyperphosphorylated, it is resistant to this degradation and accumulates within the cell . Some studies have found that increased levels of RCAN1 result in a concomitant increase in the phosphorylation of Tau and thus may contribute to its neuronal accumulation [67, 117] and we showed an accumulation of hyperphosphorylated Tau in the brains of aged RCAN1-TG mice . This observed enhancement in Tau phosphorylation may be due to the effect of RCAN1 on GSK-3 activity, since increased RCAN1 expression in PC-12 cells resulted in an increase in the absolute level of GSK-3β, which in turn enhanced its ability to phosphorylate Tau . There have also been suggestions that excess RCAN1 can exacerbate AD-like neuropathology by inhibiting calcineurin. Calcineurin activity is decreased in AD  and hyperphosphorylated tau protein and cytoskeletal changes in the brain similar to those observed in AD accumulate when the phosphatase activity of calcineurin is reduced . Thus, if RCAN1 is behaving as a calcineurin inhibitor it is possible that increased levels of RCAN1, as occurs in DS and AD, promote the development of AD  .
RCAN1, via its role as an inhibitor of calcineurin, has also been implicated in the pathogenesis of Huntington’s disease (HD). In a mouse model of HD, phosphorylation of huntingtin at serine residue 421 was protective and treatment of HD neuronal cells with calcineurin inhibitors prevented their death by maintaining their phosphorylation status at Ser421 . RCAN1-1L protein was significantly down regulated in human HD post mortem brains and exogenous expression of RCAN1-1L in a cell culture model of HD protected the cells against toxicity caused by mutant huntingtin . This protection was attributed to the ability of excess RCAN1 to inhibit calcineurin phosphatase activity, indicating that in this circumstance RCAN1 over-expression is advantageous.
Another connection between RCAN1 and neurodegeneration may be through the formation of aggregates. When proteins accumulate within a cell a mitrotubule-based apparatus known as an aggresome acts to sequester proteins within the cytoplasm. The formation of aggresomes within cells is most likely a defence mechanism against the presence of misfolded or abnormal proteins. However if these misfolded proteins are not cleared appropriately it can lead to abnormal protein accumulation and eventual neurotoxicity . The formation of aggresomes is believed to contribute to many neurodegenerative disorders including AD, Huntington’s disease and cerebral ataxia . When RCAN1 was over-expressed in various neuronal cell lines and in primary neurones, formation of aggregates occurred  and the aggregates were associated with microtubules, indicating that they had formed inclusion bodies within the cells. When RCAN1 was aggregated within neurones, neuronal abnormalities characterised by a decreased number and density of synapses were observed, which in turn altered synaptic function . This constitutes another example of the damaging effects of excess RCAN1.
Finally, two polymorphisms located in the
7. The consequences of RCAN1 over-expression in the DS immune system
7.1. The Down syndrome immune system
DS is associated with a multitude of immune system defects. People with DS are more susceptible to infections, particularly respiratory tract infections with pneumonia one of the major causes of early death . The incidence of viral hepatitis and haematopoietic malignancies is also increased in people with DS as is their tendency to develop certain types of autoimmune disorders such as autoimmune thyroid disease (AITD) (Hashimoto type), coeliac disease and diabetes  . Thus, DS appears to include a combination of immunodeficiency and immune dysfunction. Although the precise cause of this immune dysfunction is unclear, the DS immune system is characterised by a number of abnormalities thought to originate from defective innate and adaptive immunity.
7.2. Impairments in innate immunity
Innate immunity is the body’s first line of defence against invasion. This arm of the immune system either prevents the entry of pathogens into the body, or upon entry, eliminates them before they can cause any damage or disease. If a pathogen is able to gain entry into the body, innate immunity includes various non-specific mechanisms which can eliminate and destroy foreign invaders. These mechanisms include phagocytosis and inflammation. DS is associated with defects in the innate immune system. For example, natural killer (NK) cells, components of the innate immune system involved in the recognition and elimination of bacteria, viruses and tumour cells, are defective in DS individuals . Also, neutrophils from DS people exhibited a decreased ability to phagocytose  and the ability of DS-derived neutrophils and monocytes to migrate towards a site of injury or infection in response to chemokine release was reduced .
7.3. Impairments in adaptive immunity
T cell development and maturation occurs within the thymus. Bone marrow (BM) derived precursor cells migrate into the thymus where they receive developmental cues from the thymic microenvironment. Here they progress through a number of different stages of development broadly defined by the expression of CD4 and CD8 on the cell surface. Once cells become fully mature, expressing only CD4 or CD8 on the surface, they are able to migrate to the periphery and populate the immune system. The DS immune system is characterised by a number of abnormalities thought to originate from defective T cell development in the thymus. Typically, the DS thymus is small and morphologically abnormal. It exhibits cortical atrophy, loss of cortico-medullary demarcation and lymphopenia due to a defect in the development of thymocytes . The number of cells expressing high levels of the TCR α-β-CD3 complex is reduced  as are the numbers of helper (CD4+) T (Th) cells resulting in the inversion of the normal CD4+/CD8+ ratio in favour of the CD8+ population. Th cells can be further subcategorised into either Th1 or Th2 cells where Th1 cells participate in the elimination of intra-vesicular pathogens, including bacteria and parasites via the activation of macrophages, while Th2 cells clear extracellular pathogens and toxins by assisting antibody production in B cells. There is an imbalance in the T helper responses of DS individuals, although there is some disagreement as to whether it is an alteration in the Th1 or Th2 phenotype. Some studies have suggested that Th2 responses are augmented in DS based on the observation that there is an increased number of circulating CD3+/CD30 Th2 lymphocytes . Others report an increase in the Th1 population in DS and this has been attributed to increased IFNγ production  because IFNγ polarises Th0 cells towards the Th1 phenotype. While there is no doubt that a defect in T cell development and maturation within the DS thymus exists, altered apoptosis of lymphocytes may also contribute to the decrease in overall numbers of T cells in the periphery, as well as to the alterations observed in the abundance of the various T cell subsets. For example, DS CD3+ T cells and CD19+ B cells expressed significantly higher levels of early apoptotic markers compared with control cells .
T lymphocytes isolated from people with DS are also functionally compromised. Under conditions designed to simulate an infection using anti-CD3 antibodies or the non-specific mitogen, phytohemagglutinin, to activate T cells, DS lymphocytes were diminished in their proliferative capacity [136, 137]. Not only did the DS-derived T cells have a proliferative defect, they showed increased expression of apoptotic markers including APO-I/Fas (CD95) antigen, a T cell death marker, and increased apoptosis was demonstrated in cultured T cells using Annexin V . CD8+ or cytotoxic T lymphocytes (CTLs) isolated from DS individuals were also compromised in their ability to kill target cells , indicating a functional defect in this cell type also. DS-derived T cells also produce abnormal levels of cytokines, the small proteins produced by immune cells that are involved in signalling and controlling immune responses. IL-2 is central to the proliferation and differentiation of T cells and is produced by T lymphocytes once activated. Inhibition or reduction of IL-2 results in suppression of the immune system. One study on adults with DS found that the levels of IL-2 secreted from cultured stimulated T cells were significantly reduced compared with T cells cultured from normal individuals . Other studies have suggested that IL-2 is produced at comparable levels in both DS and normal individuals, but in DS the response to IL-2 may be defective . Levels of IFN-γ and TNF α are also altered in DS and although the number of DS studies is small, the consensus is that IFN- γ and TNF α levels are increased  .
In addition to T cell lymphopenia, DS individuals have marked B lymphopenia [143-145]. As well as a reduction in the number and proportions of B lymphocytes, there is a skewing of the B cell subpopulations, suggesting that maturation of B cells is defective in DS  akin to the situation with T cells, although the exact nature of this defect has not been explored. Immunoglobulin levels in DS are also abnormal, with DS B lymphocytes producing lower levels of IgM, IgG2 and IgG4 and higher levels of IgG1 [146, 147]. IgG3 and IgA levels were unchanged. Also suggesting a B cell functional deficit is the finding that antibody responses to a variety of antigens are low in DS, including the responses to pneumococcal and bacteriophage ØX174 antigens and to vaccine antigens such as tetanus, influenza A and polio [148-150].
7.4. RCAN1 in innate immunity
There is evidence to indicate that RCAN1 has a role in innate immunity and inflammation. For example, when human mononuclear cells were activated with
Importantly, RCAN1 also mediates inflammatory responses
Other studies on the role of RCAN1 in innate immunity have focussed on identifying the mechanisms by which RCAN1 regulates inflammation. One plausible means is by modulation of the NFκB signal transduction pathway. As described earlier, RCAN1 is able to inhibit NFκB signalling by increasing the stability of IκB protein . Given that NFκB is a transcription factor that controls the expression of pro-inflammatory genes and the subsequent activation of innate immune cells, negative regulation of this pathway by RCAN1 would result in inhibition of inflammation. Such a proposition is consistent with published
7.5. RCAN1 in adaptive immunity
The first evidence to suggest that RCAN1 functions in adaptive immunity came from experiments investigating T cell responses in human Jurkat cells, an immortalised T lymphocyte cell line. When these cells were stimulated with the T cell mitogens, CD3 and CD28, expression of
In addition to its function in T cells, RCAN1 is involved in the normal function of mast cells. Mast cells are specialised immune cells that contain granules rich in histamine and heparin and are known to play a role in wound healing, defence against pathogens and the pathology of IgE-dependent allergic disease and anaphylaxis . Mast cells are activated through the high affinity IgE receptor (FcεRI) on their cell surface and this activation is controlled by a number of activating and inhibitory molecules. The down regulation of mast cell activity by inhibitory signals is essential in preventing allergic disease and anaphylaxis . RCAN1 is believed to be one of these inhibitory signals. Evidence to suggest this comes from experiments conducted on RCAN1-KO mice, which displayed an exaggerated mast cell response. While RCAN1-KO mice displayed normal mast cell maturation, many of the signalling pathways following mast cell activation were perturbed. For example, mast cells isolated from RCAN1-KO mice and stimulated with FcεRI had an increase in the activation of both the NFAT and NFκB signalling pathways. As expected, there was also an increase in the expression of many pro-inflammatory genes regulated by these two pathways including
Eosinophils, another immune cell type, are predominant effector cells in allergic asthma and their presence in the lungs of asthma sufferers is regarded as a defining feature of this inflammatory disease. Absence of
8. The consequences of RCAN1 over–expression on the incidence of solid tumours in DS
8.1. Down syndrome and cancer
Individuals with DS are more likely to develop certain malignancies, especially of the immune system. There is a well-established link between leukaemia and DS, with an increased incidence in DS compared with the general population. Large population based studies conducted in different countries around the world have consistently found that the rates of leukaemia were between 10- to 19-fold higher in people with DS in comparison with the average population and there was an increased incidence of both lymphoid and myeloid leukaemias [140, 161-163]. While the incidence of both acute myeloid leukaemia (AML) and acute lymphatic leukaemia (ALL) was significantly higher in DS subjects than expected in the general population, there were significantly more cases of AML compared to ALL in DS . This increased risk is most evident at a younger age, however remained throughout life. There is also a significant increase in the incidence of neoplastic disorders such as megakaryoblastic leukaemia, where the incidence is increased about 500-fold in DS [164, 165]. In males, there is also a link between DS and testicular cancer, possibly due to higher levels of follicular stimulating hormone, hypogonadism or cryptorchidism [166, 167]. Notably, those with DS are less likely to develop other solid tumours such as neuroblastomas and breast and lung cancers [162, 163, 168]. Indeed, individuals with DS had a 50% reduction in the incidence of solid tumours compared to the number of cases expected in the general population and this was observed over all age groups examined . Thus it seems likely that a number of tumour suppressor genes reside on
8.2. RCAN1 and tumourigenesis
While the identities of the
The strongest genetic evidence to suggest a role for
Finally, the significance of RCAN1 in tumour suppression in DS was elegantly demonstrated using yet another DS genetic model. TS65Dn mice that harbour a third copy of many
In this review we have attempted to summarise what is currently known about the function of the