Expansion of outer cortical CUX2 neurons requires adaptations for DNA repair | Nature

## Summary
Scale bar, 100 μm. g , h , Cux2 mRNA ISH in P7 Emx1-Cre;Atf4 fl/fl and control cortices ( g ) and quantification of Cux2 + neurons within a 500 × 500-μm 2 cortical region ( h ) ( n = 4 mice per genotype). Scale bar, 100 μm. i , j , Immunostaining of CUX1 and CUX2 in P7 Emx1-Cre;Atf4 fl/fl and control cortices ( i ) and quantification of CUX1 and CUX2 double-positive neurons within a 500 × 500-μm 2 cortical region ( j ) ( n = 4 mice per genotype). Scale bar, 100 μm. k , l , CTIP2 immunostaining in P7 Emx1-Cre;Atf4 fl/fl and control cortices ( k ) and quantification of CTIP2 + neurons in layers 2/3/4, 5 and 6 within a 500 × 500-μm 2 cortical region ( l ) ( n = 4 mice per genotype). Scale bar, 100 μm. m , n , TBR1 immunostaining in P7 Emx1-Cre;Atf4 fl/fl and control cortices ( m ) and quantification of TBR1 + neurons within a 500 × 500-μm 2 cortical region ( n ) ( n = 4 mice per genotype).

## Article Content
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Subjects
Neural progenitors
Neuronal development
Abstract
During mammalian evolution, excitatory neurons in upper cortical layer 2 and layer 3 (L2/3) have shown a disproportionate expansion compared with other layers
1
,
2
,
3
,
4
. Replicative expansion of cortical neural progenitors is associated with considerable oxidative DNA damage. Here we show that activating transcription factor 4 (ATF4) has roles as a critical regulator of the DNA damage response, directly activating components of double-stranded DNA repair, including CIRBP, UBA52 and EBF1. Notably, pan-cortical knockout (
Emx1-Cre;
Atf4
fl/fl
) demonstrates that ATF4 is required specifically for the development of upper layer 2/3 neurons, marked by the expression of cut-like homeobox 2 protein, CUX2. ATF4 functions to repair DNA damage and attenuate cell death of embryonic radial glial progenitors in a p53-dependent manner. In particular, we show that cold inducible RNA-binding protein (CIRBP) is a transcriptional target of ATF4 that is required for normal phosphorylation of the key double-strand DNA repair factor ataxia telangiectasia mutated (ATM). These findings establish that ATF4 is an essential regulator of the DNA damage response. They further indicate that there are extraordinary requirements for DNA repair after replicative stress in CUX2
+
neurons during mammalian brain development.
Main
Corticogenesis during mammalian embryonic development requires a massive expansion of neural progenitors (NPs) and their subsequent development into the mature neuron subtypes that make up the six neocortical layers
1
. During mammalian evolution, upper cortical L2/3 show a disproportionate expansion compared with other layers
2
, implying that there are higher-order functions as well as increased replicative stress during development. In the mature cortex, these upper cortical layers (L2/3) are occupied predominantly by pyramidal projection neurons that send and receive mainly cortico-cortical connections
3
,
4
, and the extent and complexity of these interhemispheric connections is closely associated with higher cognitive functions in mammals
5
. CUX2 is a CUT-homeodomain transcription factor that is expressed during early development in a subset of cortical neuronal progenitors, and remains an identity marker of these upper L2/3 projection neurons in the mature cortex
6
,
7
. It has been suggested that these L2/3 neurons are fate restricted, even in early cortical development, and that a radial glial (RG)-cell lineage is intrinsically specified to generate only CUX2
+
upper-layer neurons independent of niche and birth date
8
, but this has been contested
9
,
10
. Several studies now suggest that these L2/3 CUX2
+
cortical neurons are fundamentally affected in a multitude of human disorders. Loss of CUX2
+
neurons has been reported in multiple sclerosis
11
,
12
, head trauma in young human athletes
13
, Alzheimer’s disease
14
and frontotemporal dementia
15
. Dysfunction of L2/3 neurons is also consistently linked to temporal lobe epilepsy
16
and schizophrenia
17
, and they show most pathway dysregulation in human autism
18
. This indicates an intrinsic vulnerability to various types of central nervous system dysfunction. Important aspects of CUX2
+
neuron basic biology, and the reason for this vulnerability, are not understood.
During corticogenesis, the rapid hyperproliferation of NPs in the subventricular zone demands a huge energy supply, and the reactive oxygen species (ROS) generated by mitochondrial respiration and cellular metabolism
19
are a main cause of DNA damage in proliferating NPs
20
. The preservation of genome stability is crucial
21
,
22
,
23
, because transmission of genetic errors during early progenitor expansion leads to a variety of severe neurodevelopmental neurological conditions and neurodegeneration. Eukaryotic cells, including NPs, have evolved a finely tuned signalling network called the DNA damage response (DDR)
24
,
25
, which detects DNA damage and then integrates cell-cycle control with DNA repair, or triggers apoptosis through p53-induced cell-cycle arrest in cells in which the DNA cannot be repaired. The DDR has evolved repair mechanisms specific for many types of DNA lesion, the most common of which during neurogenesis are DNA double-strand breaks (DSBs)
26
. ATM is a central regulator of DNA DSB repair and, once activated by DSBs, it phosphorylates many downstream factors to initiate the cascade of double-strand repair
27
. Phosphorylation of ATM at serine 1981 is required for the sustained retention of ATM at DSBs, and also for its ability to phosphorylate its downstream targets after DNA damage
28
. However, important gaps remain in our understanding of the factors that govern neural-progenitor genome integrity, and a more complete appreciation of how DNA damage-signalling pathways promote neural development is needed to understand the formation of the human brain.
ATF4 is a multifunctional transcripti

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## Expert Analysis

### Merits
- Important aspects of CUX2 + neuron basic biology, and the reason for this vulnerability, are not understood.
- However, important gaps remain in our understanding of the factors that govern neural-progenitor genome integrity, and a more complete appreciation of how DNA damage-signalling pathways promote neural development is needed to understand the formation of the human brain.
- P < 0.0001 ( b ), P < 0.0001 ( d , left), P < 0.0001 ( d , middle), P < 0.0001 ( d , right), P = 0.000003 ( f , layer 2/3/4), P = 0.210781 ( f , layer 5/6), P < 0.0001 ( h ), P < 0.0001 ( j ), P = 0.24373 ( l , layer 2/3/4), P = 0.125173 ( l , layer 5), P = 0.062794 ( l , layer 6), P = 0.1003 ( n ), P = 0.000002 ( p , layer 2/3/4), P = 0.545881 ( p , layer 5/6), P < 0.0001 ( r ), P < 0.0001 ( t ), P = 0.5465 ( v ), P = 0.1662 ( x ), P = 0.8374 ( z ); NS, not significant.

### Areas for Consideration
N/A

### Implications
N/A

### Expert Commentary
This article covers cre, neurons, dna topics. Notable strengths include discussion of cre. Readability: Flesch-Kincaid grade 0.0. Word count: 2614.