Temporal downregulation of the polyubiquitin gene Ubb affects neuronal differentiation, but not maturation, in cells cultured in vitro

Abstract

Reduced levels of cellular ubiquitin (Ub) pools due to disruption of the polyubiquitin gene Ubb lead to dysregulation of neural stem cell (NSC) differentiation and impaired neuronal maturation in cells isolated from Ubb−/− mouse embryonic brains. However, it is currently unknown whether Ub is required for the specific stage of neuronal development or whether it plays a pleiotropic role throughout the process. To answer this question, we aimed to downregulate Ubb expression temporally during neuronal development, which could not be achieved in Ubb −/− cells. Therefore, we exploited lentivirusmediated knockdown (KD) of Ubb at different stages of neuronal development, and investigated their phenotypes. Here, we report the outcome of Ubb KD on two independent culture days in vitro (DIV): DIV1 and DIV7. We observed that NSCs did not differentiate properly via Ubb KD on DIV1, but the maturation of already differentiated neurons was intact via Ubb KD on DIV7. Intriguingly, Ubb KD activated Notch signaling when it had been suppressed, but exerted no effect when it had already been activated. Therefore, our study suggests that Ub plays a pivotal role in NSC differentiation to suppress Notch signaling, but not in the subsequent maturation stages of neurons that had already been differentiated.Ubiquitin (Ub) is one of the most abundant eukaryotic proteins involved in post-translational modifications 1-3 . Ubiquitylation of target substrates occurs via the actions of three enzymes: E1 Ub-activating enzymes, E2 Ub-conjugation enzymes, and E3 Ub ligases 4,5 . The fate of the substrates is determined by the type of ubiquitylation, i.e., monoubiquitylation or polyubiquitylation with a specific Ub chain linkages 6,7 . The best-known and most important outcome is the polyubiquitylation of substrates with Lys 48 (K48) linkages and their targeting to the 26S proteasome for degradation 8,9 . A timely degradation of substrates is also important for the differentiation of neural stem cells (NSCs), onset of neurogenesis, neuronal development, and neuronal function 10,11 . Indeed, reduced levels of cellular Ub via the disruption of the polyubiquitin gene Ubb compromised the degradation of substrates and resulted in the dysregulation of NSC differentiation with inhibition of neurogenesis and impaired neuronal maturation [12][13][14] . NSC differentiation is mainly regulated by Notch signaling 15,16 . Typically, Notch signaling is suppressed to promote neurogenesis during embryonic stages, while it is activated to promote gliogenesis and neuronal maturation during postnatal stages [17][18][19][20] . Notch signaling is initiated by the interaction between the Notch receptor and its ligand Delta (DLL1) in the neighboring cells, followed by the cleavage and release of Notch intracellular domain (NICD) via γ-secretase. NICD then translocates into the nucleus to form a transactivator complex and activates the transcription of target genes 21 . Notch target genes such as the Hairy/enhancer of split (Hes) and Hairy/enhancer-of-split related with...

Lipocalin-2: a therapeutic target to overcome neurodegenerative diseases by regulating reactive astrogliosis

“…Ub deficiency also appears to upregulate Lcn2 expression in mixed neuronal cells 118 . This may be due to a higher percentage of GFAP-positive astrocytes in Ubb KO or knockdown (KD) cells than in wild-type cells.…”

Reversible Regulation of Polyubiquitin Gene UBC via Modified Inducible CRISPR/Cas9 System

“…Before reverse transcription, RNA samples were treated with DNase I (amplification grade; Invitrogen, Carlsbad, CA, USA) for 15 min at room temperature. Conventional and quantitative RT-PCR (qRT-PCR) were performed as previously described [45]. Briefly, RNA concentration was measured, and 1 μg of total RNA was used as a template for reverse transcription using SuperiorScript II Reverse Transcriptase (#RT005; Enzynomics, Daejeon, Korea), and 1/20 of the resulting cDNA was used as template for conventional and qRT-PCR.…”

“…Cells were lysed in RIPA buffer and immunoblot analysis was performed as previously described [45]. Briefly, total cell lysates (15 μg) were subjected to SDS-PAGE, followed by immunoblot detection with the appropriate antibodies.…”

Regulation of polyubiquitin genes to meet cellular ubiquitin requirement

“…In fact, in neural stem cells (NSCs) with free Ub levels reduced because of Ubb knockout or knockdown, the degradation of the Notch intracellular domain (NICD) is delayed, leading to the activation of Notch signaling and upregulating the expression of Notch target genes ( 35 , 36 ). Activation of Notch signaling in the early stages of neuronal development suppresses neurogenesis and promotes premature gliogenesis, causing abnormal differentiation of NSCs, degeneration of defective neurons, and reactive gliosis ( 35 ).…”

“…To answer this question, ectopic Ub overexpression has been used to increase Ub levels ( 37 , 57 , 58 ). Conversely, a method used to reduce Ub levels is a knockdown through shRNA or use of cells isolated from knockout mice ( 36 - 38 , 59 , 60 ). These methods may alter the Ub levels whatever are the expression levels of endogenous genes, although overexpressed Ub has been shown to downregulate the expression of polyubiquitin genes.…”