Compromised astrocyte function and survival negatively impact neurons in infantile neuronal ceroid lipofuscinosis

Jenny Lange,Brenda P. Williams,Jennifer M. Pocock,Mark S. Sands,Emyr Lloyd-Evans,Luke J. Haslett,Jonathan D. Cooper

doi:10.1186/s40478-018-0575-4

Abstract

The neuronal ceroid lipofuscinoses (NCLs) are the most common cause of childhood dementia and are invariably fatal. Early localized glial activation occurs in these disorders, and accurately predicts where neuronal loss is most pronounced. Recent evidence suggests that glial dysfunction may contribute to neuron loss, and we have now explored this possibility in infantile NCL (INCL, CLN1 disease). We grew primary cultures of astrocytes, microglia, and neurons derived from Ppt1 deficient mice (Ppt1−/−) and assessed their properties compared to wildtype (WT) cultures, before co-culturing them in different combinations (astrocytes with microglia, astrocytes or microglia with neurons, all three cell types together). These studies revealed that both Ppt1−/− astrocytes and microglia exhibit a more activated phenotype under basal unstimulated conditions, as well as alterations to their protein expression profile following pharmacological stimulation. Ppt1- /− astrocytes also displayed abnormal calcium signalling and an elevated cytoplasmic Ca2+ level, and a profound defect in their survival. Ppt1−/− neurons displayed decreased neurite outgrowth, altered complexity, a reduction in cell body size, and impaired neuron survival with prolonged time in culture. In co-cultures, the presence of both astrocytes and microglia from Ppt1−/− mice further impaired the morphology of both wild type and Ppt1−/− neurons. This negative influence was more pronounced for Ppt1−/− microglia, which appeared to trigger increased Ppt1−/− neuronal death. In contrast, wild type glial cells, especially astrocytes, ameliorated some of the morphological defects observed in Ppt1−/− neurons. These findings suggest that both Ppt1−/− microglia and astrocytes are dysfunctional and may contribute to the neurodegeneration observed in CLN1 disease. However, the dysfunctional phenotypes of Ppt1−/− glia are different from those present in CLN3 disease, suggesting that the pathogenic role of glia may differ between NCLs.

Highlights

The neuronal ceroid lipofuscinoses (NCLs, or Batten disease) are a large group of inherited lysosomal storage disorders, each caused by mutations in an individual gene [8, 32]
Over 99% of DAPI positive cells in astrocyte cultures of either genotype were positive for the astrocyte marker glutamine synthetase (GS) (WT: 99.2% GS + ve, 0.71% CD68 + ve, and 0% O4 + ve; Ppt1−/−: 99.6% GS + ve, 0.35% CD68 + ve, and 0% O4 + ve)
Under basal conditions the morphology of cultured Ppt1−/− astrocytes was more heterogeneous than that seen in wild type (WT) cultures, with many GFAP-positive astrocytes appearing larger in Ppt1−/− cultures (Fig. 1A)

Summary

Introduction

The neuronal ceroid lipofuscinoses (NCLs, or Batten disease) are a large group of inherited lysosomal storage disorders, each caused by mutations in an individual gene [8, 32]. Despite recent advances in the treatment of late infantile NCL (CLN2 disease) via enzyme replacement therapy [30], the other forms of NCL all remain untreatable This includes infantile NCL (INCL or CLN1 disease), an earlier onset and more rapidly progressing form of NCL, which is caused by a mutation in the CLN1/PPT1 gene (Vesa et al, 1995; [5, 21]). It remains unclear how deficiency in palmitoyl protein thioesterase-1 (PPT1), the lysosomal enzyme encoded by this gene results in the devastating neurodegenerative impact upon the brain that typifies CLN1 disease

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Results

Conclusion