Deficiency of prosaposin/saposins results in incomplete catabolism of GSLs and their age dependent accumulation in the brain [7
]. To understand the molecular changes at the transcriptome level during the disease course of prosaposin deficiency, three mouse models were compared in this study by microarray analysis: 1) a complete deficiency [PS-/-], 2) a hypomorph [PS-NA], and 3) a hypomorph together with a GCase mutation [4L/PS-NA]. In these three chronic neurological disease models, significant numbers of genes were up- or down-regulated at birth prior to any behavioral, histological or significant GSL abnormalities. The differentially expressed genes included those involved in transcription and signal transduction that were early molecular responses to the insult from incomplete GSL degradation. With age, GSL (Table ) accumulation became detectible and the surrounding astrocytes and microglial cells sensed the insult and promoted proinflammatory responses to maintain tissues homeostasis. The alterations of proinflammatory genes were common in all three models, which reflect the generalized distribution of microglial cells and astrocytes in the brain. Prolonged proinflammatory reactions also are present in other neurodegenerative diseases [19
]; they can promote beneficial effects, i.e., repair, as well as detrimental or destructive effects in the brain. The loss of irreplaceable neurons in PS-NA and 4L/PS-NA mice started at ~12 wks. The negative TUNEL assays [7
] indicated an alternative cell death pathway, e.g., necrosis or autophagy, in these mouse brains. Importantly, neurobehavioral impairments in PS-NA and 4L/PS-NA mice preceded detectible neuronal cell loss, suggesting that the functional impairments preceded the structural deficits, and that the accumulating (even subclinical) GSL had direct effects on overall cell regulation. The basis for the underlying neurological deficits might be attributed to synaptic dysfunction as has been found in ataxia and Alzheimer's disease [21
]. Alteration of synaptic and proteosome genes in the current models implicates GSL storage in the lysosome as affecting synaptic function and leading to dendritic retraction and neuronal death. As a potential link to this pathogenic mechanism, ubiquitin proteases that are linked to synaptic activity [22
] were altered in these models. Differential expression of the ubiquitin protease genes was identified in PS-/-, PS-NA and 4L/PS-NA mouse brains and accumulation of ubiquitin was found in the brains of all three mouse models (Sun, et al., unpublished observation). Clarification is needed for the interaction between ubiquitin-dependent protein turnover, and the plastic and degenerative changes at synapses in brains with GSL accumulation.
Among the differentially expressed genes, CEBPD was the only transcription factor up-regulated in the cerebella and cerebra of all three mouse models. This unique commonality was not obscured by the potential diluting effect from the combined analyses of sub-regions of cerebrum or genetic background variation between 4L/PS-NA (FVB/C57BL/129) and PS-/-, PS-NA and WT (FVB). Network analysis indicated that CEBPD directly or indirectly connects to gene functional effector pathways. For example, CEBPD modulates GFAP and expression of numerous cytokines through FOS mediated proinflammatory pathways (Fig. ). CEBPD also modulates MBP and FOS expression. MBP interacted with myelin-associated genes and both MBP and FOS connected into the cell death pathways. CEBPD has been previously implicated in peripheral immune challenge [24
] and CEBPD is up-regulated after traumatic brain injury in rats and in brains from patients with Alzheimer's disease [25
]. Lipopolysaccharide (LPS) and galactosylsphingosine are exogenous ligands that regulate CEBPD expression in visceral tissues and astrocytes [27
]. In response to LPS treatment, CEBPD and NF-kB are recruited to the CNS [32
]. Galactosylsphingosine, a cytotoxic metabolite of galactosyl ceramide found in Krabbe disease, induces expression of cytokine mediated nuclear translocation of CEBPD [28
]. The present data suggest that accumulated GSLs in prosaposin deficient brains might be endogenous lipids that mediate CEBPD expression. Furthermore, CEBPD binds to cytokine promoters in macrophages and astrocytes [33
]. The expression of CEBPD is mediated by Sp1, STAT3, c-Rel and c-Jun [35
], whereas the downstream genes MBP and GFAP, as well as several inflammatory genes, are modulated by CEBPD in astrocytes [30
CEBPD is widely expressed in the PNS and CNS. CEBPD in combination with CREB (cyclic AMP-responsive element-binding protein) participates in nerve growth factor gene transcription [38
]. Curiously, the CEBPD knockout mice have no major phenotypes except enhanced contextual fear, whereas, CEBPA and CEBPB knockouts exhibit perinatal lethality or rapid deterioration within months after birth [39
]. Such findings suggest that CEBPD is not essential in non-stressed physiological development, but may be important in response to pathological insults by playing a role in the modulation of proinflammatory responses and neuronal homeostasis in neurodegenerative diseases. CEBPD expression was up-regulated after 10 days in PS-/- mice and after 4 wks in PS-NA and 4L/PS-NA mice. The temporal change of this expression correlated directly with the biochemical and histological progression of the disease. Apparently, CEBPD plays a role in the CNS of prosaposin deficiency mice in promoting disease progression.
Atf3 has been suggested as an "adaptive response" gene by responding to extra- and intra-cellular changes induced by stress signals and signals for promoting cell proliferation [42
]. Atf3 is induced by LPS and is a negative regulator of TLR 4 and Ccl4 released from macrophages [43
]. Atf3 has been suggested as having a role in controlling gene expression programs required for axon regeneration [45
]. The increase of Atf3 might be involved in an adaptive response to the disease in the present models. Nfia promotes gliogenesis in spinal cord [46
] and directs the differentiation of cerebellar granular neurons [47
]. Disruption of Nfia in mice leads to perinatal lethality, agenesis of the corpus callosum, and abnormal formation of midline glial structures [12
]. Both Atf3 and Nfia were present in the CEBPD network, suggesting potential interactions effects between these transcription factors.
Gene expression profiles have been studied in several lysosomal diseases. By serial analysis of gene expression (SAGE), many up-regulated genes are attributed to the proinflammation pathways in the GM2
gangliosidoses, Tay-Sachs and Sandhoff disease patients' brains [49
]. In the severe neuronopathic Gaucher disease type 2, with increased GC, the genes in brain associated with microglial cells and astrocytes are slightly elevated and the neuronal genes has small depressions [50
]. Using a cDNA microarray, human Niemann-Pick C1 fibroblasts revealed oxidative stress, impairment of trafficking and regulation of calcium in response to abnormal cholesterol and GSL accumulation [51
]. This is quite different from that obtained in our prosaposin deficient mice. The transcription profiles demonstrated that molecular changes involved proinflammation, trafficking, and loss of neuron cells. These results suggest a GSL and cellular specificity in response to the disease. Interestingly, several lysosomal enzymes showed up-regulation (β-glucuronidase, α-mannosidase and β-hexosaminidase A and B) in the present models, suggesting altered lysosomal function compensating for GSL storage.