Microglia-Specific Promoter Activities of HEXB Gene

Abstract

Adeno-associated virus (AAV)-mediated genetic targeting of microglia remains a challenge. Overcoming this hurdle is essential for gene editing in the central nervous system (CNS). Here, we characterized the minimal/native promoter of the HEXB gene, which is known to be specifically and stably expressed in the microglia during homeostatic and pathological conditions. Dual reporter and serial deletion assays identified the critical role of the natural 5’ untranslated region (−97 bp related to the first ATG) in driving transcriptional activity of the mouse Hexb gene. The native promoter region of mouse, human, and monkey HEXB are located at −135, −134, and −170 bp to the first ATG, respectively. These promoters were highly active and specific in microglia with strong cross-species transcriptional activities, but did not exhibit activity in primary astrocytes. In addition, we identified a 135 bp promoter of CD68 gene that was highly active in microglia but not in astrocytes. Considering that HEXB is specifically expressed in microglia, these data suggest that the newly characterized microglia-specific HEXB minimal/native promoter can be an ideal candidate for microglia-targeting AAV gene therapy in the CNS.

Established and emerging techniques for the study of microglia: visualization, depletion, and fate mapping

“…Although HEXB immunostaining has been utilized in combination with TMEM119 to directly identify microglia ( Jia et al, 2021 ), Hexb -based gene reporter and fate mapping tools, have also recently provided important insights into the behavior and functions of microglia in vivo ( Masuda et al, 2020 ). Additionally, the promoter region of Hexb has recently been characterized, enabling the study of Hexb as a potential target for microglia-targeted gene therapies in humans ( Shah et al, 2022 ).…”

“…Recent studies using RNAscope and single-cell RNA sequencing have shown that Hexb is expressed in brain microglia and its expression is highly restricted to these cells within the CNS (Masuda et al, 2020;Faust et al, 2023). Notably, it was not detected in CNS-associated macrophages according to Masuda et al (2020); however, it has been identified in non-CNS cells, such as Ly6C + monocytes obtained from the bone marrow (Hickman et al, 2013;Monaghan et al, 2019;Masuda et al, 2020;Shah et al, 2022). HEXB may also play a role in pathology, with elevated gene expression of HEXB associated with poor prognosis in human glioblastoma (Jia et al, 2021).…”

Infiltrating Peripheral Monocyte TREM-1 Mediates Dopaminergic Neuron Injury in Substantia Nigra of Parkinson's Disease Model Mice

“…To evaluate the activation status of innate immune cells in PD model mice, we rst examined the resident and in ltrating Mo/MΦs in the SNpc. Recent massive single-cell analyses revealed that Hexb is exclusively expressed in brain microglia but not in Mo/MΦs [37,40]. Importantly, this newly de ned microglia-speci c gene identi ed Hexb as a stably expressed microglial core gene during homeostasis and disease, Iba1 is a calcium-binding protein both expressed in microglia and Mo/MΦs.…”

Emerging Techniques for the Study of Microglia: Visualization, Depletion and Fate Mapping

“…Similarly, Hexb is a lysosomal enzyme that play critical role in lysis processes, breaking down fatty compounds such as sphingolipids and gangliosides (Kuil et al, 2019). Recent studies using RNAscope and single-cell RNA sequencing have shown that Hexb is exclusively expressed in brain microglia but not in monocytes/macrophages across homeostasis and pathological conditions in mice (Hickman et al, 2013;Masuda et al, 2020;Shah et al, 2022). HEXB may also play a role in pathology, with elevated gene expression of HEXB associated with poor prognosis in human gliobastoma (Jia et al, 2021).…”

“…Although HEXB immunostaining has been utilized in combination with TMEM119 to directly identify microglia (Jia et al, 2021), Hexb-based gene reporter and fate mapping tools, have also recently provided important insights into the behavior and functions of microglia in vivo (Masuda et al, 2020) (discussed below). Additionally, the promoter region of Hexb has recently been characterized, enabling the study of Hexb as a potential target for microglia-targeted gene therapies in humans (Shah et al, 2022).…”