Increasing evidence supports the idea that glia of all types, including astrocytes, oligodendrocytes, and microglia, each of which has close contact with neurons, help support, in various ways, the neighboring neurons. For example, astrocytes, the major cellular component of the central nervous system (CNS), play important roles in synapse formation and plasticity, and in preventing neuronal excitotoxicity by rapid removal of excess glutamate through glutamate transporters 33,34
. Thus, it is perhaps not surprising that many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia (SCA), Huntington’s disease, Parkinson’s disease and multiple system atrophy (MSA), were recently shown to have an astrocytic component. In these disorders, mutant products in astrocytes and microglia damage neighboring neurons, either by release of toxic components or by mutant-mediated reduction in neuronal support functions 29
. Astrocytes can also affect neurons indirectly. For example, multiple sclerosis (MS), which is caused by oligodendrocyte degeneration, is initiated and progressed in part by astrocytes expressing toxic compounds, which then damage oligodendrocytes, leading to impaired neuronal signaling 35,36
RTT is distinguished from these other neurodegenerative or neurological disorders in being initiated by loss of MeCP2 function rather than by gain of function of a toxic mutant protein. While previous studies focused on MeCP2 loss-of-function in brain and specifically in neurons, the effects in non-neuronal cells in the brain were generally overlooked. Earlier in vivo experiments, however, suggested that further studies were warranted: (i) Gene expression profiles of postmortem female RTT brain revealed decreased levels of expression of neuronal genes encoding synaptic markers and increased levels of expression of glial genes involved in neuropathological mechanisms 37
, and (ii) MRI and MRS studies showed that not only neuronal but also glial metabolism was affected in RTT mouse brain 38,39
. Despite these changes, obvious neuronal and glial degeneration had not been reported in Rett Syndrome 40
, and the balance between neuronal and glial lineages produced from neural progenitors appeared normal 21
. Further, the amounts of GFAP in different regions of wild-type and RTT brains, as well as in astrocytic cultures from RTT and wild-type mice, are indistinguishable from each other (Supplementary Fig. S1
and data not shown), indicating that the number of astrocytes in RTT and wild-type brains is similar. These observations suggest that Rett Syndrome is not caused by reduced numbers but rather by dysfunction of specific cell types in the brain. Nonetheless, unlike mutant neurons, studies addressing the direct involvement of mutant glia in the neuropathology of Rett Syndrome have been lacking, in part due to the uncertainty of the presence of MeCP2 in glia.
Our studies show that MeCP2 is expressed not only in neurons, but also in all types of glia of normal adult brain, while it is absent in glia of RTT brain. Importantly, our co-culture studies show that astrocytes from RTT male mice, as well as their conditioned medium, cause aberrant dendritic morphology in both mutant and wild-type neurons, which resemble hippocampal pyramidal and granule cell abnormalities in conventional RTT male animals in vivo
. This suggests that, in female human Rett Syndrome patients, who are mosaic for loss of MeCP2 function, wild-type neurons are likely to be affected in a non-cell autonomous fashion by the mutant astrocytes. Supporting this notion is the finding that in heterozygous human patients, the majority of pyramidal cortical neurons show aberrant dendritic morphology 41
. Furthermore, in culture, both RTT and wild-type neurons survive and extend processes in the presence of conditioned medium from wild-type, but not mutant astrocytes. This observation suggests that, consistent with in vivo
studies, the damage to mutant neurons is not irreversible 20
and thus potentially can be rescued by therapeutic intervention.
While aberrant dendritic morphology was the predominant effect of MeCP2-null astrocytes on the neurons, at least to some extent, neuronal survival was also affected. Although it is generally accepted that Rett Syndrome is not a neurodegenerative disorder, several earlier studies suggest that some neurodegeneration occurs in human RTT 42,43
. Further studies are required to address more systematically whether mild neuronal degeneration occurs, at least in some circumstances, in RTT patients.
The astrocytic effect could be due to depletion of a molecule essential for neuronal dendritic morphology or to a soluble secreted factor that is detrimental to neurons. For example, depletion of neurotrophic factors such as the glial-cell-line-derived neurotrophic factor, GDNF, which affects dendritic branching, or molecules secreted from glia with deleterious effects such as tumor necrosis factor-alpha (TNFα) and nitric oxide (NO), could cause aberrant morphology and/or loss in neuronal functions. By screening for several gene candidates whose aberrant expression could potentially perturb the levels of such essential molecules, we found that the expression of the branched-chain aminotransferase (BCAT) mRNA was up-regulated by 3-fold in MeCP2-null relative to wild-type astrocytic cultures. BCAT catalyzes the transamination of branched chain amino acids, the nitrogen donor for synthesis of glutamate in the brain, and thus can modulate the supply of glutamate. Further biochemical studies will determine whether a toxic factor is secreted from the mutant astrocytes. In this case, identification of the aberrantly secreted factor(s) could ultimately provide a means of pharmacological intervention for RTT.
Astrocytes in RTT animals could damage neurons through different non-cell autonomous pathways. It could be that: (i) astrocytes are not affected directly by loss of MeCP2 function, but the mutant neurons stimulate damaging responses from glia that then affect the neurons; (ii) astrocytes are affected directly by loss of MeCP2 and this is the primary source of neurotoxicity; (iii) both astrocytes and neurons are affected directly by loss of MeCP2, but loss of MeCP2 from glia causes a glial damage response that enhances the initial damage in neurons. The later scenario has precedence in ALS; although mutant SOD1 expression in motor neurons is required for disease initiation, neurotoxicity is additionally produced by damage within the neighboring mutant glia, which facilitates the initiation and progression of the disease 44,45
. Further in vivo
studies are required to distinguish between these possible mechanisms. Towards this end, we are currently generating mouse models in which MeCP2 null astrocytes are produced in a background of wild type neurons. Preliminary findings suggest that loss of MeCP2 selectively in astrocytes elicits, at least in part, a RTT-like phenotype, including decreased body weight and hindlimb clasping, supporting the notion that MeCP2-dysfunction in glia contributes to the neuropathology of Rett Syndrome.