A serious challenge in elucidating the pathogenesis of human HPRT deficiency is its rarity, with so few patients or patient-derived materials available for experimental investigations. A further challenge in elucidating the mechanisms responsible for the neurological abnormalities is the relative inaccessibility of relevant brain pathways to experimental investigations. In view of these challenges, surrogate experimental models have been used for exploring pathogenesis. In the current studies, we took advantage of multiple independent cell models for HPRT deficiency. Because of the central importance of dopamine neuron dysfunction to LND, we focused on the 10 recently developed HPRT−
sublines of the dopaminergic mouse MN9D neuroblastoma line (23
). These cells replicate the key phenomena of significant dopamine loss without gross baseline morphological defects or impaired viability evident in Lesch-Nyhan human brains and HPRT−
mice. A microarray survey of these cell models pointed to abnormalities in pathways relevant to early neuronal development and differentiation. Rigorous and methodical evaluation by qPCR revealed abnormal expression of 15 of 29 mRNAs known to encode proteins relevant to dopamine neuron development and differentiation. The most robust quantitative changes involved overexpression of the engrailed genes (En1
) and their products (engrailed 1 and 2). The changes in the developmental markers do not reflect a global and non-specific abnormality affecting all mRNAs in the HPRT−
MN9D sublines, since multiple control mRNAs were normal.
While cell models have some obvious advantages as experimental tools for identifying novel mechanisms of pathogenesis, they also have some important limitations. One limitation of cell models is that individual sublines of established cell lines may exhibit idiosyncratic properties that differ from the parental controls independent of the genetic alteration introduced (22
). This limitation was first addressed in the current studies by evaluating 10 independently isolated HPRT−
MN9D sublines. The statistical approach for the microarray and qPCR studies involved comparing the parental MN9D cell line against all 10 HPRT−
MN9D sublines, searching for significant changes across the whole group of mutant sublines, rather than idiosyncratic changes among individual sublines. This approach reduces the risk of finding spurious abnormalities unrelated to HPRT deficiency and revealed highly significant changes in multiple developmentally regulated mRNAs, most notably over-expression of the engrailed genes. The relationship between HPRT deficiency and over-expression of the engrailed genes was further tested by examining the influence of restoring functional HPRT in the HPRT−
MN9D sublines in which En1
were over-expressed. The over-expression of the engrailed genes was partly or completely reversed in each case. Thus, the increased expression of engrailed is not a spurious phenomenon in the mutant sublines, but one that appears to depend on the expression of functional HPRT.
Another limitation of cell models is that findings may be idiosyncratic to the cell model studied, with no relevance to other experimental models. This limitation was addressed by evaluating HPRT− cell models from multiple different sources. In addition to the 10 HPRT− MN9D sublines, we examined four HPRT− sublines of human M17 neuroblastoma. All four of the mutant M17 sublines exhibited an increase in En1 expression, while En2 expression was more variable. We also evaluated primary human fibroblasts from eight patients with varying degrees of HPRT deficiency. There was again evidence for increased expression of En1 and/or En2 in the HPRT− fibroblasts relative to controls. Thus, increased expression of one or both engrailed genes appears to be common to several cell models of HPRT deficiency, including those derived from affected patients.
The final limitation of cell models is that the findings may be restricted to the in vitro
environment, with little relevance for the in vivo
state, especially in the brain. This limitation can be addressed only by examining brain tissue from affected Lesch-Nyhan patients. Because the developmental markers in question have specific temporal windows of expression, it may be necessary to study these brains at various stages of development. Though such studies are not feasible because of the absence of appropriate human brain specimens, it is important to note that the hypothesis regarding a defect in developmental programming of the dopamine neurochemical phenotype is consistent with prior human studies showing a relatively broad disruption of many biochemical markers of the dopamine phenotype (7
) with no apparent histological evidence for loss of cells or axonal projections (2
A disruption of molecular pathways that control early development provides a novel mechanism that could account for the well-known association between HPRT deficiency and dysfunction of brain dopamine systems. The En1
genes encode two closely related transcription factors that are part of a large family of homeobox transcription factors responsible for many developmental processes. The engrailed products have specific roles in two events during neural development. Early in embryogenesis, they take part in establishing the size and region of the midbrain in which dopamine neurons later develop. Later they play a role in the specification and survival of dopamine neurons (28
). They function as nuclear transcription factors that alter the expression of multiple other genes, but they also can be released from cells and internalized by neighbors, where they are thought to exert paracrine-like activities (31
). The genes have partly overlapping functions, such that one can partly compensate for loss of the other (33
). In mutant mice homozygous for null mutations in both genes, dopamine neurons are generated between embryonic days 11 and 14, but the entire population disappears before birth and the pups cannot survive (28
). Intermediate genotypes with one or more copies of either gene display varying degrees of midbrain dopamine neuron loss (24
). In our HPRT−
MN9D sublines, there were no apparent correlations between the expression of En1
among the HPRT−
cells. Some HPRT−
cells exhibited an increase in both engrailed genes, while others exhibited increases in only one. The lack of consistent increases in both mRNA and protein products may be related to the redundant biological functions of these two homeoproteins.
There is good evidence that alterations in the expression of the engrailed genes influence dopamine neuron development and survival. Transcription factors of the homeoprotein family present cell autonomous and non-cell autonomous activities that can influence neurite elongation and guidance. In the case of engrailed, several of its putative transcriptional targets may play such a function. Among the latter targets are EphrinA5, a cell surface protein with collapsing activity (35
) and MAP1B a microtubule associated protein that regulates microtubule polymerization in the axon (36
). More recently, it was shown that engrailed serves as an axon guidance factor, in a non-cell autonomous way, by regulating mRNA translation within the growth cones (37
). It is also of interest that engrailed 1 is present in the dendrites of midbrain dopaminergic neurons (38
). Although the latter localization is in want of a function, the ability of engrailed to regulate translation in developing axons leaves open the possibility that a similar mechanism is used to regulate dendrite geometry. Thus, the morphological defects seen in our differentiated HPRT−
MN9D cells might represent downstream consequences of abnormal expression of the engrailed.
The mechanisms by which HPRT deficiency influences the expression of the engrailed genes remain to be determined. One possibility is that engrailed expression may be affected directly by HPRT. While there currently is no evidence that HPRT can affect the engrailed or other developmental pathways directly, there is ample precedent for other housekeeping enzymes having such additional ‘moonlighting’ activities in addition to their traditionally ascribed functions (39
). Thus, HPRT may have a moonlighting function beyond its role in purine recycling. Alternatively, HPRT deficiency may influence engrailed pathways indirectly, through the secondary changes in purine metabolism that occur when HPRT is missing. Indeed, disruption of purine levels is known to have an important influence on neuronal differentiation in other cells (42
). It also is possible that over-expression of engrailed genes reflects a compensatory developmental response to a defect elsewhere in the developmental programs of these neurons. Whether the effect is direct or indirect, the implication of the current findings is that HPRT-mediated purine recycling has an important role in regulating early developmental programming of dopaminergic neurons. Further studies will be required to more completely delineate the complex cascade of events resulting from HPRT deficiency and their influence on brain development.