Written by - Qin Shangyao

Editor-in-charge - Wang Sizhen

Editor — Binwei Yang

Topoisomerases (Topoisomerase) are a highly conserved and widely distributed class of proteins, genetically located on chromosome 17q21-22, molecular weight of 170 kD, is a nuclear content second only to histone proteins biological macromolecules

Adult Neurogenesis is conserved among mammals and plays a very important role

Over the past few decades, the interaction between SVZ exogenous and intrinsic factors has been shown to regulate the activity of NSCs in adult neurogenesis, but the underlying mechanisms remain largely unknown

On August 27, 2022, Professor He Cheng and Professor Su Zhida of naval military medical university published a study entitled "Topoisomerase IIA in adult NSCs regulates SVZ neurogenesis by transcriptional activation of Usp37" at the biomedical journal Nucleic Acids Research.

Multiple studies have shown that TOP2a can regulate the progress

Figure 1 Expression patterns of TOP2a in the developmental stage and adult brain

(Source: Qin S, et al.

Subsequently, using the method of in vitro culture of adult NSCs (Figure 2A), the authors found that the use of interfering RNA and specific inhibitors for TOP2a can effectively block the self-replication ability of adult NSCs, which is manifested in the decline of the number of neurospheres and proliferation potential in vitro (Figure 2B-H
).

Through in vitro inhibition experiments, the authors preliminarily confirmed the need
for TOP2a for adult neurogenesis.

Figure 2 Self-renewal of adult neural stem cells in vitro requires the participation of TOP2a

(Source: Qin S, et al.
, Nucleic Acids Res, 2022)

To verify the effect of TOP2a on adult NSCs in vivo, the authors established a CreER-Loxp conditioned knockout mouse model for the TOP2a gene (Figure 3A) and found that the model was effective in reducing the expression of TOP2a at both mRNA and protein levels (Figures 3B, C
).

However, when the authors tried to examine the model's effect on SVZ adult NSCs, they found that this did not significantly change their overall number (Figures 3D, E
).

Given the expression pattern of TOP2a and the proportion of activated NSCs, the researchers further examined the effects of the model on resting NSCs and activated NSCs, respectively, and found that knockout of TOP2a only reduced the number of activated NSCs (Figure 3F-I
).

Fig.
3 Response of adult neural stem cells after knockout of TOP2a in vivo

(Source: Qin S, et al.
, Nucleic Acids Res, 2022)

In order to further illustrate that the deletion of TOP2a only affects the activation of adult NSCs in vivo, the authors used concentrated lentiviral injection to interfere with the expression of TOP2a at SVZ, and found that this can significantly inhibit the proliferative activity of adult NSCs here (Figure 4A-F
).

For the SVZ adult neurogenesis process, resting NSCs will be further specialized into two main types of intermediate precursor cells (transient expansion precursor cells, TAPs, and neuroblasts) after activation, which express unique molecular markers Ascl1 and DCX
, respectively.

Using a conditionally induced mouse model, the authors found that TOP2a deletion significantly reduced the number of two major precursor cells (Figure 4G-J
).

Using the results of Figures 3 and 4, the authors demonstrate the necessary role
of TOP2a for the normal production of nerve cells at SVZ and for the maintenance of adult neurogenesis.

Figure 4 Cell proliferation and intermediate precursor cell response after knockout of TOP2a in vivo

(Source: Qin S, et al.
, Nucleic Acids Res, 2022)

Precursor cells of DCX+ are child cells produced by activated NSCs in SVZ, which can migrate to the distal olfactory sphere (OB) via the RMS tangent, and then from the GRANULOCYTE layer (GCL) of the OB radially migrating to the oligosphere (GL) of the OB, eventually differentiating into interneurons and integrating into the neural circuits
of the olfactory bulb.

Therefore, the researchers further examined whether the conditioned knockout of TOP2a in adult NSCs ultimately affects the neurogenesis
of OB.

The authors first used BrdU markers to migrate from SVZ to OB of nascent neurons (Figure 5A), and found that knockout of TOP2a significantly reduced the number of newborn neurons at OB (Figure 5B-E), which eventually led to a change in the number of mature OB interneurons (BrdU+/NeuN+) (Figures 5F, G).


In addition, the authors also examined the subtypes of interneurons affected by TOP2a, and found that TOP2a knockout mainly reduced the two types of interneurons at THE OB at CR+ and CB+ (Fig.
5H-I)
by co-infecting the marker of each interneuron.

This phenomenon shows that TOP2a not only affects the production of neural precursor cells at SVZ, but also plays a key role
in maintaining specific types of neurogenesis at OB.

Fig.
5 In vivo knockout TOP2a affects adult neurogenesis at olfactory bulbs

(Source: Qin S, et al.
, Nucleic Acids Res, 2022)

In order to dig deeper into the underlying molecular mechanism of THET2a action in adult neurogenesis, the researchers down-regulated the expression of TOP2a in adult NSCs using a specific inhibitor (PluriSin#2) to analyze RNA-Seq (Figure 6A, B
).

Through differential expression analysis, gene function annotation, and pathway analysis, the authors found that the treatment group could significantly inhibit the cell cycle activity, transcriptional activity, and expression of stem cell-related genes of adult NSCs compared with the control group, and promote the expression of differentiation-related genes (Figure 6C-F
).

These data provide preliminary molecular evidence that TOP2a plays a key role in the proliferation of adult NSCs, indicating that the downregulation of TOP2a is not conducive to maintaining its stem cell properties
.

Fig.
6 Inhibition of transcriptomics changes in adult NSCs after TOP2a

(Source: Qin S, et al.
, Nucleic Acids Res, 2022)

To identify possible TOP2a target genes in adult NSCs, the authors also performed genomic ChIP-Seq analysis using C-terminal domain-specific antibodies of TOP2a (Figure 7), which can distinguish the binding region
of its homologous isomeric molecule TOP2b.

Through binding site analysis, the authors found that the genes bound to TOP2a were mainly related to Notch, Wnt, hippocampal signaling pathways, and were mainly localized in the introton, intergene, and promoter regions of genes (Figures 7A-C
).

Through de novo Motif analysis, the authors screened out two stem cell function-related Motifs that could bind to transcription factors, including Tcf12, Ascl1, Sox2, and Sox3, suggesting that TOP2a may regulate the activity of NSCs in a transcription-dependent manner (Figures 7D, E).


Next, the authors identified 8 candidate TOP2a direct-acting target genes by integrating RNA-Seq and ChIP-Seq, among which Usp37, Birc6, Ddx51 and Gna14 were associated with cell cycle regulation.
Sox2, Hes5, Nup153, and Nup133 are associated with stem cell properties, and these target genes have been validated by ChIP-qPCR and sequencing results (Figures 7F, G
).

In the end, the authors determined that the most likely downstream target molecule for TOP2a was USP37 (Figure 7H) through experimentally validated methods
.

Figure 7 Use ChIP-Seq and RNA-Seq to look for potential downstream target genes for TOP2a

(Source: Qin S, et al.
, Nucleic Acids Res, 2022)

USP37 is a member of a newly discovered family of ubiquitin-specific proteases that hinder ubiquitin-mediated degradation of target proteins
.

It mainly promotes the conversion
of G1/S phases by mediating the deubiquitination of protein molecules CYCLIN-A (including CCNA1 and CCNA2) as an effective regulator of the cell cycle.

In addition, the USP37 regulates DNA replication
by stabilizing CDT1.

The authors found that under the conditions of TOP2a knockdown, the expression of USP37 and its downstream target protein decreased synchronously, and this effect could be partially reversed by upregulating USP37, which further demonstrated that USP37 is the direct target gene of TOP2a (Figure 8A-E
).

At the same time, the decrease in the number and size of neurospheres caused by TOP2a inhibition can also be restored to a considerable level by upregulating USP37 (Figure 8F, G
).

Finally, by injecting the overexpressed lentivirus of USP37 at the site, the authors found that the inhibition of adult neurogenesis in conditioned knockout mice was also reversed to some extent (Figures 8H, I
).

In summary, these data suggest that USP37 plays a role as a direct target gene for TOP2a, and can participate in maintaining the proliferative activity
of adult NSCs under the regulation of TOP2a.

Fig.
8 Overexpression of USP37 can reverse the inhibitory effect of TOP2a knockout on adult neurogenesis

(Source: Qin S, et al.
, Nucleic Acids Res, 2022)

In summary, given that previous studies on topoisomerase II.
A (TOP2a) have focused almost exclusively on cell cycle regulation and tumorigenesis, researchers have studied the expression, function, and regulation mechanisms
of this molecule for the first time in the field of adult neurogenesis.

This study helps people to fully understand the molecular function of TOP2a, and also provides a new molecular regulation mechanism for the proliferation, self-renewal and differentiation of adult neural stem cells, and provides new ideas and theoretical basis
for the clinical use of stem cells to treat neurological damage, degeneration and other diseases.

Of course, it is worth noting that most of the enrichment peaks in TOP2a's ChIP-Seq experiments are located in intergenes or introns, suggesting that the regulatory function of TOP2a may also be related
to mechanisms such as conformation of chromatin and 3D genome folding.

TOP1 and TOP2b have been reported to coordinate transcriptional activation
through the proximal pause mechanism of the RNA polymerase II promoter and enrichment of DSBs.

Therefore, other regulatory pathways may have contributed to the role of TOP2a in the transcription process, and these problems require further in-depth study to solve
.

Link to the original article: https://doi.
org/10.
1093/nar/gkac731

Lecturer Qin Shangyao of the School of Basic Medical Sciences of the Naval Military Medical University is the first author, Lecturer Yuan Yimin is the co-first author, and Professor He Cheng and Professor Su Zhida are the corresponding authors
of this article.

The research was supported
by the Brain Program of the Ministry of Science and Technology of China (2022ZD0204702), the National Natural Science Foundation of China (81971161, 82171386, 31871026, 82002012), the Shanghai Science and Technology Development Fund (22YF1458600), the Naval Military Medical University Fund (2021QN08) and the Major Project of the Shanghai Municipal Ministry of Science and Technology (No.
2018SHZDZX01).

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