Biochemical and Morphological Characterization of a Neurodevelopmental Disorder-Related Mono-ADP-Ribosylhydrolase, MACRO Domain Containing 2

Abstract

MACRO Domain Containing 2 (MacroD2) is a neurodevelopmental disorder-related mono-ADP-ribosylhydrolase. Molecular features of this protein in neural tissues are largely unknown. In this study, we generated a specific antibody against MacroD2, and carried out expression and morphological analyses of the molecule during mouse brain development. In Western blotting, 2 MacroD2 isoforms with molecular masses of ∼70 and ∼75 kDa started to be expressed at embryonic day 16.5, reached the maximal level at postnatal day 8, and then gradually decreased through P30. In contrast, other isoforms with molecular masses of ∼110 and ∼140 kDa gradually increased during embryonic to postnatal development. In immunohistochemical analyses, MacroD2 was strongly detected in cortical neurons in layer II–V at P0 and P7, while the protein expression decreased significantly in the neurons at P30. Immunofluorescence analyses revealed that MacroD2 was mainly distributed in the soma and to a lesser extent in the axon and dendrite of immature primary cultured mouse hippocampal neurons. On the other hand, in the matured hippocampal neurons, while MacroD2 was detected in the soma, it displayed in dendrites a punctate distribution pattern with a partial colocalization with synaptic markers, synaptophysin, and PSD95. The obtained results indicate that MacroD2 is expressed and may have a physiological role in the central nervous system during brain development.

(ADP-ribosyl)hydrolases: structure, function, and biology

“…These mostly pathoneurological phenotypes of the MacroD2 gene are associated with its loss of function, thus suggesting a physiological role in the central nervous system. This correlates with robust neuronal expression of MacroD2 during brain development (Ito et al 2018).…”

Comparative analysis of MACROD1, MACROD2 and TARG1 expression, localisation and interactome

“…This is larger than the predicted molecular weight of the canonical MACROD2 isoform and might represent effects of particular amino acid sequences or posttranslational modification(s). A recent study revealed specific expression of MacroD2 in cortical and hippocampal neurons in the mouse brain 50 , agreeing with our findings of relatively high MACROD2 expression in human neuroblastoma cells. Together, these data show that the three hydrolases are expressed in different tissues.…”

The Controversial Roles of ADP-Ribosyl Hydrolases MACROD1, MACROD2 and TARG1 in Carcinogenesis

“…However, it was not tested whether the identified compound might inhibit one of the macrodomains with catalytic activity [69]. [40,70,71] Ubiquitous expression, enriched in skeletal muscle [70,71] mRNA expression is induced by oestrogen [72][73][74] MACROD2 C20orf133 MACROD2 47 kDa 50 kDa [70] Diffuse nuclear, cytoplasmic [70,75] So far detected only in the brain [70,76] Phosphorylation by ATM upon DNA damage induces translocation to cytoplasm [75] TARG1 C6orf130 OARD1 17 kDa 17 kDa [14,70] Nuclear, nucleolar, stress granular [14,45,70] Ubiquitous expression [70] Leaves nucleoli upon DNA damage [14] * The predicted molecular weight of MACROD1 is 35 kDa. However, due to cleavage of the N-terminus upon translocation into mitochondria, the protein detected in western blotting (WB) is smaller [70].…”

“…TARG1 and MACROD1 are expressed throughout the different tissues with an enrichment of MACROD1 in skeletal muscle at both the protein and RNA level [70]. MACROD2 has, thus far, only been detected in human neuroblastoma cells and in mouse cortical neurons [70,76]. Several reports have correlated polymorphisms or deletions within the MACROD2 gene with autism-spectrum disorders [77][78][79][80].…”

“…Several reports have correlated polymorphisms or deletions within the MACROD2 gene with autism-spectrum disorders [77][78][79][80]. It is not clear, however, whether the MACROD2 gene product itself or surrounding genes are responsible for this association even though elevated protein expression in neurons supports a potential brain-specific function of MACROD2 [76]. However, a long non-coding RNA has been identified within an intron of the MACROD2 gene, which is more highly expressed in most tissues investigated than the MACROD2 mRNA, and, thereby, potentially confuses correlations of mutations in the MACROD2 gene and phenotypes [81].…”