BACKGROUND
degeneration of axonal and/or synaptic compartments of neurons is an early and pathologically important process in many disorders of the human nervous system, ranging from alzheimer's disease and batten disease through to multiple sclerosis and motor neuron disease  <cit> .
therapies designed to specifically delay or halt the progression of axonal and synaptic degeneration are therefore actively being sought for a wide range of neurological disorders.
the most robust delay in axonal and synaptic degeneration reported to date in animal models of neurological disorders has been generated by the introduction of the slow wallerian degeneration  gene.
to date, the wlds gene has been shown to significantly modify disease onset and/or progression in animal models of traumatic axonal injury  <cit> , parkinson's disease  <cit> , demyelinating neuropathies  <cit> , some forms of motor neuron disease  <cit>  and cerebral ischemia  <cit> .
these experiments highlight the potential for using the wlds protein and/or its downstream molecular interactions to generate novel therapeutic approaches for the treatment of neurological disorders.
importantly, the ability to successfully deliver the wlds gene and confer robust neuroprotection using gene therapy approaches  <cit>  has opened up the possibility of directly delivering wlds-related therapies to human patients.
the chimeric wlds gene occurred as the result of a spontaneous mutation in the c57bl/ <dig> line of mice , resulting in a tandem triplication of a region already present on the distal region of chromosome  <dig>  mice carrying the wlds mutation are otherwise indistinguishable from their c57bl/6j strain mates in genotyping of more than  <dig> microsatellite markers and restriction fragment length polymorphisms .
the triplicated region contains sequences coding for nmnat <dig>  rbp <dig> and ube4b  <cit> .
the boundaries within the triplicated region result in  <dig> copies of a fusion gene comprising the n <dig> terminal amino acids of ube4b and the entire coding region of nmnat <dig> , linked by  <dig> amino acids from the 5' untranslated region of nmnat <dig> which are not normally expressed  <cit> .
the chimeric portion of the triplication  has been shown to be sufficient to recapitulate the wlds phenotype through the generation of transgenic lines in mice, rats and drosophila  <cit> .
although the wlds gene has obvious therapeutic potential, its mechanism of action remains unclear.
however, several studies have shown that expression of wlds protein in neurons in vivo and in vitro induces changes in core cellular pathways, including; nad biosynthesis, ubiquitination, the mitochondrial proteome, cell cycle status and cell stress  <cit> .
even though the extent to which each of these modifications contribute to the neuroprotective phenotype remains unclear, the fact that wlds modifies key cellular pathways raises potential problems for its use as a therapeutic agent that have yet to be investigated.
for example, it is conceivable that changes in nad biosynthesis pathways and/or cell cycle pathways in non-neuronal organs and tissues may alter their form and/or function.
here, we have undertaken the first detailed study of the effects of wlds expression in non-neuronal tissues in vivo.
we show that wlds protein is expressed at differing levels in a range of non-neuronal organs in wlds mice.
systemic expression of wlds did not affect overall body weight or growth.
gravimetric and histological analysis of a wide range of organs and tissues confirmed no changes in wlds mice.
we also demonstrate that previously reported alterations in cell cycle and cell stress proteins reported in wlds brain tissue are a neuronal-specific response not observed in non-neuronal organs in vivo.
RESULTS
wlds expression is not limited to neuronal tissue in vivo
despite numerous reports in the literature concerning the therapeutic potential of the wlds gene , no previous studies have undertaken a rigorous assessment of the consequences of wlds expression in non-neuronal tissue.
we first examined the expression of wlds protein and rna levels in a variety of organs from wlds mice and wild-type controls and compared expression levels to those observed in the cerebellum  <cit> .
organs examined included liver, kidney, heart, lung and spleen.
we used quantitative fluorescent western blotting techniques to assess and compare protein expression levels using a proven antibody for the wlds protein .
example bands are shown in figure 1a for liver , spleen  and the cerebellum .
these experiments showed that wlds protein expression was not limited to neuronal tissue, but that it was not strongly expressed in all organs examined .
for example, the liver had the lowest expression at  <dig>  ±  <dig>   fold increase compared to wild-type background signal, the spleen showed a  <dig>  ±  <dig>  fold increase, whereas the cerebellum showed a  <dig>  ±  <dig>  fold increase.
we also examined rna expression in wlds mice  and found that rna expression varied greatly between different organs and did not necessarily correlate with protein expression.
for example, the liver  had relatively high rna expression levels, approaching those observed in the cerebellum.
taken together, these findings demonstrate that wlds protein and rna are present in a range of non-neuronal organs at differing levels, but also demonstrate that the presence of rna does not always indicate that wlds protein will also be present at comparable levels.
the observation that wlds protein is strongly expressed in non-neuronal tissues led us to investigate whether there would be sufficient protein in tail tips to facilitate accurate genotyping of wlds mice using western blotting.
we found that quantitative western blotting techniques easily distinguished between wild-type, heterozygous and homozygous wlds mice .
systemic expression of wlds did not affect gross appearance or growth rates
in order to test whether systemic wlds expression affected total body weight, mice were bred from parents heterozygous for the wlds mutation .
litters from this heterozygote cross contained mice which were null for wlds , mice which were heterozygous for wlds  and mice which were homozygous for wlds .
all experimental comparisons were carried out within litters to remove any potential effects of background strain.
there were no obvious qualitative differences in the size or behaviour of mice within litters.
figure 3a shows  <dig> female mice from the same experimental litter , indistinguishable from one another.
mice were weighed at regular intervals post weaning up to  <dig> months of age.
there were no significant differences between the body weights of wt, het and wlds mice at any time-point examined .
wlds expression did not affect gravimetrics or histopathology of non-neuronal tissue
detailed necropsies were carried out by an experienced rodent pathologist  on wt, het and wlds littermates to determine if non-neuronal expression of wlds protein had any affects on tissue gravimetrics  or histopathology .
organs examined for gravimetrics included kidneys, liver, spleen, thymus, heart and whole brain.
in order to minimize the effects of natural biological variability within litters data were expressed as organ weight per grams/body weight.
no significant differences were observed between mice from any of the  <dig> genotypes .
mice were also examined for alterations in body fat deposits , with no evident differences .
histopathological assessments were carried out on the following organs and tissues: kidney, heart, lungs, mediastinum, liver, gallbladder, urinary bladder, vagina, uterus, ovary, spleen, pancreas, lymph nodes, salivary gland, pituitary gland, adrenal gland, stomach, intestines , calvarial bone, femur, tibia, knee joint, spine and spinal cord , eyes, head, various sympathetic and parasympathetic ganglia, brain .
no significant differences were observed between any of the genotypes.
example sections for comparison are shown in figure  <dig>  figure 4a shows representative images of liver from wild-type and wlds mice, a tissue with little to no detectable wlds protein expression .
note the presence of normal hepatocyte nuclei, sinusoids and central veins in both images.
figure 4b shows representative images of kidney cortex, a tissue with intermediate levels of wlds protein expression, showing the presence of normal glomeruli, bowman's capsules and cortical tubular profiles.
figure 4c shows representative images of spleen, a tissue with high wlds protein expression, with normal red pulp and white pulp lymphoid accumulations.
cell cycle and cell stress alterations are specific to neuronal tissue
we have previously reported alterations in proteins involved with cell cycle and cell stress in non-injured neural tissue from wlds mice in vivo and in vitro  <cit> .
in order to determine whether similar changes were instigated in non-neuronal organs and tissues expressing wlds protein we examined expression levels of the cell stress marker antiphosphohistone h2ax  and a marker of cell cycle progression acetylated histone h <dig>  in liver, kidney, heart, lung, spleen and cerebellum from wild-type and wlds mice.
example bands for both of these proteins are shown in figure 5a.
as previously demonstrated there was a significant  increase in expression of both cell stress and cell cycle markers in wlds cerebellum compared to wild-type .
however, no changes in expression of either of these proteins were found in any of the non-neuronal organs examined .
thus, previously reported differences in cell cycle and cell stress status appear to be a neuronal-specific response to expression of wlds at a basal level.
discussion
here we have shown that wlds rna and protein are expressed at differing levels in a range of non-neuronal organs in wlds mice but did not influence overall body weight or growth.
gravimetric and histological analysis of a wide range of organs demonstrated that the presence of wlds rna and/or protein had no overt influence on non-neuronal tissues at a basal level.
we have also shown that previously reported alterations in cell cycle and cell stress proteins reported in wlds brain tissue are neuronal-specific and are not observed in non-neuronal organs in vivo.
it will now be of interest to establish whether wlds protein can modify responses to injury in non-neuronal tissues and organs, where it is expressed.
expression data in the current study show that the spleen has a comparatively high level of protein, suggesting that this organ may be useful for such studies.
these data demonstrate that downstream consequences of wlds expression, incorporating pathways including nad biosynthesis, ubiquitination, the mitochondrial proteome, cell cycle status and cell stress  <cit> , do not have any major adverse effects on non-neuronal organs.
this suggests that the utilization of wlds-based therapeutics for treating neurodegenerative conditions can be considered safe for other body systems, should the treatment spread beyond the confines of the nervous system .
studies reporting that wlds-mediated neuroprotection  can be successfully delivered to the nervous system using approaches such as viral gene delivery suggest that wlds-based therapeutics are a realistic possibility  <cit> .
our experimental data have also revealed nervous system-specific effects of wlds expression.
the finding that modifications in cell cycle and cell stress status previously reported in neurons expressing wlds  <cit>  are not replicated in non-neuronal tissues suggests that neurons may have distinct intrinsic responses to the presence of wlds.
one possible explanation for this is that neurons are terminally differentiated cells whose cell cycle status is markedly different from many non-neuronal cells .
whether or not this contributes directly to the neuroprotective phenotype remains to be determined.
nevertheless, these data support the hypothesis that wlds acts by targeting a specific step in degenerative pathways intrinsic to neurons  <cit> .
CONCLUSIONS
we conclude that expression of wlds protein has no adverse effects on non-neuronal tissue at a basal level in vivo, supporting the possibility of its safe use in future therapeutic strategies targeting axonal and/or synaptic compartments in patients with neurodegenerative disease.

 degeneration of axonal and/or synaptic compartments of neurons is an early and pathologically important process in many disorders of the human nervous system, ranging from alzheimer's disease and batten disease through to multiple sclerosis and motor neuron disease. therapies designed to specifically delay or halt the progression of axonal and synaptic degeneration are therefore actively being sought for a wide range of neurological disorders. the most robust delay in axonal and synaptic degeneration reported to date in animal models of neurological disorders has been generated by the introduction of the slow wallerian degeneration gene. to date, the wlds gene has been shown to significantly modify disease onset and/or progression in animal models of traumatic axonal injury, parkinson's disease, demyelinating neuropathies, some forms of motor neuron disease and cerebral ischemia. these experiments highlight the potential for using the wlds protein and/or its downstream molecular interactions to generate novel therapeutic approaches for the treatment of neurological disorders. importantly, the ability to successfully deliver the wlds gene and confer robust neuroprotection using gene therapy approaches has opened up the possibility of directly delivering wlds-related therapies to human patients. the chimeric wlds gene occurred as the result of a spontaneous mutation in the c57bl/ line of mice, resulting in a tandem triplication of a region already present on the distal region of chromosome mice carrying the wlds mutation are otherwise indistinguishable from their c57bl/6j strain mates in genotyping of more than microsatellite markers and restriction fragment length polymorphisms. the triplicated region contains sequences coding for nmnat rbp and ube4b. the boundaries within the triplicated region result in copies of a fusion gene comprising the n terminal amino acids of ube4b and the entire coding region of nmnat, linked by amino acids from the 5' untranslated region of nmnat which are not normally expressed. the chimeric portion of the triplication has been shown to be sufficient to recapitulate the wlds phenotype through the generation of transgenic lines in mice, rats and drosophila. although the wlds gene has obvious therapeutic potential, its mechanism of action remains unclear. however, several studies have shown that expression of wlds protein in neurons in vivo and in vitro induces changes in core cellular pathways, including; nad biosynthesis, ubiquitination, the mitochondrial proteome, cell cycle status and cell stress. even though the extent to which each of these modifications contribute to the neuroprotective phenotype remains unclear, the fact that wlds modifies key cellular pathways raises potential problems for its use as a therapeutic agent that have yet to be investigated. for example, it is conceivable that changes in nad biosynthesis pathways and/or cell cycle pathways in non-neuronal organs and tissues may alter their form and/or function. here, we have undertaken the first detailed study of the effects of wlds expression in non-neuronal tissues in vivo. we show that wlds protein is expressed at differing levels in a range of non-neuronal organs in wlds mice. systemic expression of wlds did not affect overall body weight or growth. gravimetric and histological analysis of a wide range of organs and tissues confirmed no changes in wlds mice. we also demonstrate that previously reported alterations in cell cycle and cell stress proteins reported in wlds brain tissue are a neuronal-specific response not observed in non-neuronal organs in vivo.  wlds expression is not limited to neuronal tissue in vivo despite numerous reports in the literature concerning the therapeutic potential of the wlds gene, no previous studies have undertaken a rigorous assessment of the consequences of wlds expression in non-neuronal tissue. we first examined the expression of wlds protein and rna levels in a variety of organs from wlds mice and wild-type controls and compared expression levels to those observed in the cerebellum. organs examined included liver, kidney, heart, lung and spleen. we used quantitative fluorescent western blotting techniques to assess and compare protein expression levels using a proven antibody for the wlds protein. example bands are shown in figure 1a for liver, spleen and the cerebellum. these experiments showed that wlds protein expression was not limited to neuronal tissue, but that it was not strongly expressed in all organs examined. for example, the liver had the lowest expression at ± fold increase compared to wild-type background signal, the spleen showed a ± fold increase, whereas the cerebellum showed a ± fold increase. we also examined rna expression in wlds mice and found that rna expression varied greatly between different organs and did not necessarily correlate with protein expression. for example, the liver had relatively high rna expression levels, approaching those observed in the cerebellum. taken together, these findings demonstrate that wlds protein and rna are present in a range of non-neuronal organs at differing levels, but also demonstrate that the presence of rna does not always indicate that wlds protein will also be present at comparable levels. the observation that wlds protein is strongly expressed in non-neuronal tissues led us to investigate whether there would be sufficient protein in tail tips to facilitate accurate genotyping of wlds mice using western blotting. we found that quantitative western blotting techniques easily distinguished between wild-type, heterozygous and homozygous wlds mice. systemic expression of wlds did not affect gross appearance or growth rates in order to test whether systemic wlds expression affected total body weight, mice were bred from parents heterozygous for the wlds mutation. litters from this heterozygote cross contained mice which were null for wlds, mice which were heterozygous for wlds and mice which were homozygous for wlds. all experimental comparisons were carried out within litters to remove any potential effects of background strain. there were no obvious qualitative differences in the size or behaviour of mice within litters. figure 3a shows female mice from the same experimental litter, indistinguishable from one another. mice were weighed at regular intervals post weaning up to months of age. there were no significant differences between the body weights of wt, het and wlds mice at any time-point examined. wlds expression did not affect gravimetrics or histopathology of non-neuronal tissue detailed necropsies were carried out by an experienced rodent pathologist on wt, het and wlds littermates to determine if non-neuronal expression of wlds protein had any affects on tissue gravimetrics or histopathology. organs examined for gravimetrics included kidneys, liver, spleen, thymus, heart and whole brain. in order to minimize the effects of natural biological variability within litters data were expressed as organ weight per grams/body weight. no significant differences were observed between mice from any of the genotypes. mice were also examined for alterations in body fat deposits, with no evident differences. histopathological assessments were carried out on the following organs and tissues: kidney, heart, lungs, mediastinum, liver, gallbladder, urinary bladder, vagina, uterus, ovary, spleen, pancreas, lymph nodes, salivary gland, pituitary gland, adrenal gland, stomach, intestines, calvarial bone, femur, tibia, knee joint, spine and spinal cord, eyes, head, various sympathetic and parasympathetic ganglia, brain. no significant differences were observed between any of the genotypes. example sections for comparison are shown in figure figure 4a shows representative images of liver from wild-type and wlds mice, a tissue with little to no detectable wlds protein expression. note the presence of normal hepatocyte nuclei, sinusoids and central veins in both images. figure 4b shows representative images of kidney cortex, a tissue with intermediate levels of wlds protein expression, showing the presence of normal glomeruli, bowman's capsules and cortical tubular profiles. figure 4c shows representative images of spleen, a tissue with high wlds protein expression, with normal red pulp and white pulp lymphoid accumulations. cell cycle and cell stress alterations are specific to neuronal tissue we have previously reported alterations in proteins involved with cell cycle and cell stress in non-injured neural tissue from wlds mice in vivo and in vitro. in order to determine whether similar changes were instigated in non-neuronal organs and tissues expressing wlds protein we examined expression levels of the cell stress marker antiphosphohistone h2ax and a marker of cell cycle progression acetylated histone h in liver, kidney, heart, lung, spleen and cerebellum from wild-type and wlds mice. example bands for both of these proteins are shown in figure 5a. as previously demonstrated there was a significant increase in expression of both cell stress and cell cycle markers in wlds cerebellum compared to wild-type. however, no changes in expression of either of these proteins were found in any of the non-neuronal organs examined. thus, previously reported differences in cell cycle and cell stress status appear to be a neuronal-specific response to expression of wlds at a basal level. discussion here we have shown that wlds rna and protein are expressed at differing levels in a range of non-neuronal organs in wlds mice but did not influence overall body weight or growth. gravimetric and histological analysis of a wide range of organs demonstrated that the presence of wlds rna and/or protein had no overt influence on non-neuronal tissues at a basal level. we have also shown that previously reported alterations in cell cycle and cell stress proteins reported in wlds brain tissue are neuronal-specific and are not observed in non-neuronal organs in vivo. it will now be of interest to establish whether wlds protein can modify responses to injury in non-neuronal tissues and organs, where it is expressed. expression data in the current study show that the spleen has a comparatively high level of protein, suggesting that this organ may be useful for such studies. these data demonstrate that downstream consequences of wlds expression, incorporating pathways including nad biosynthesis, ubiquitination, the mitochondrial proteome, cell cycle status and cell stress, do not have any major adverse effects on non-neuronal organs. this suggests that the utilization of wlds-based therapeutics for treating neurodegenerative conditions can be considered safe for other body systems, should the treatment spread beyond the confines of the nervous system. studies reporting that wlds-mediated neuroprotection can be successfully delivered to the nervous system using approaches such as viral gene delivery suggest that wlds-based therapeutics are a realistic possibility. our experimental data have also revealed nervous system-specific effects of wlds expression. the finding that modifications in cell cycle and cell stress status previously reported in neurons expressing wlds are not replicated in non-neuronal tissues suggests that neurons may have distinct intrinsic responses to the presence of wlds. one possible explanation for this is that neurons are terminally differentiated cells whose cell cycle status is markedly different from many non-neuronal cells. whether or not this contributes directly to the neuroprotective phenotype remains to be determined. nevertheless, these data support the hypothesis that wlds acts by targeting a specific step in degenerative pathways intrinsic to neurons.  we conclude that expression of wlds protein has no adverse effects on non-neuronal tissue at a basal level in vivo, supporting the possibility of its safe use in future therapeutic strategies targeting axonal and/or synaptic compartments in patients with neurodegenerative disease. 
we conclude that expression of wlds protein has no adverse effects on non-neuronal tissue at a basal level in vivo, supporting the possibility of its safe use in future therapeutic strategies targeting axonal and/or synaptic compartments in patients with neurodegenerative disease. we show that expression of wlds rna and protein are not restricted to neuronal tissue, but that the relative rna and protein expression levels rarely correlate in these non-neuronal tissues. how the chimeric wlds protein confers neuroprotection remains controversial, but several studies have shown that expression in neurons in vivo and in vitro modifies key cellular pathways, including; nad biosynthesis, ubiquitination, the mitochondrial proteome, cell cycle status and cell stress. finally, we demonstrate that previously reported wlds-induced changes in cell cycle and cell stress status are neuronal-specific, not recapitulated in non-neuronal tissues at a basal level. whether similar changes are induced in non-neuronal tissue and organs at a basal level in vivo remains to be determined. we have undertaken a detailed analysis of non-neuronal wlds expression in wlds mice, alongside gravimetric and histological analyses, to examine the influence of wlds expression in non-neuronal tissues. future experiments determining whether wlds protein can modify responses to injury in non-neuronal tissue are now required. the wlds gene encodes a novel ube4b-nmnat chimeric protein that is responsible for conferring the neuroprotective phenotype. this may be of particular importance for the development and application of neuroprotective therapeutic strategies based around wlds-mediated pathways designed for use in human patients. we show that wlds mice have normal body weight and growth characteristics as well as gravimetrically and histologically normal organs, regardless of wlds protein levels.
 the slow wallerian degeneration gene specifically protects axonal and synaptic compartments of neurons from a wide variety of degeneration-inducing stimuli, including; traumatic injury, parkinson's disease, demyelinating neuropathies, some forms of motor neuron disease and global cerebral ischemia. the wlds gene encodes a novel ube4b-nmnat chimeric protein that is responsible for conferring the neuroprotective phenotype. how the chimeric wlds protein confers neuroprotection remains controversial, but several studies have shown that expression in neurons in vivo and in vitro modifies key cellular pathways, including; nad biosynthesis, ubiquitination, the mitochondrial proteome, cell cycle status and cell stress. whether similar changes are induced in non-neuronal tissue and organs at a basal level in vivo remains to be determined. this may be of particular importance for the development and application of neuroprotective therapeutic strategies based around wlds-mediated pathways designed for use in human patients.  we have undertaken a detailed analysis of non-neuronal wlds expression in wlds mice, alongside gravimetric and histological analyses, to examine the influence of wlds expression in non-neuronal tissues. we show that expression of wlds rna and protein are not restricted to neuronal tissue, but that the relative rna and protein expression levels rarely correlate in these non-neuronal tissues. we show that wlds mice have normal body weight and growth characteristics as well as gravimetrically and histologically normal organs, regardless of wlds protein levels. finally, we demonstrate that previously reported wlds-induced changes in cell cycle and cell stress status are neuronal-specific, not recapitulated in non-neuronal tissues at a basal level.  we conclude that expression of wlds protein has no adverse effects on non-neuronal tissue at a basal level in vivo, supporting the possibility of its safe use in future therapeutic strategies targeting axonal and/or synaptic compartments in patients with neurodegenerative disease. future experiments determining whether wlds protein can modify responses to injury in non-neuronal tissue are now required. 