The Role of PAX2 in Neurodevelopment and Disease.

In developmental biology, transcription factors are involved in regulating the process of neural development, controlling the differentiation of nerve cells, and affecting the normal functioning of neural circuits. Transcription factors regulate the expression of multiple genes at the same time and have become a key gene category that is recognized to be disrupted in neurodevelopmental disorders such as autism spectrum disorders. This paper briefly introduces the expression and role of PAX2 in neurodevelopment and discusses the neurodevelopmental disorders associated with Pax2 mutations and its possible mechanism. Firstly, mutations in the human Pax2 gene are associated with abnormalities in multiple systems which can result in neurodevelopmental disorders such as intellectual disability, epilepsy and autism spectrum disorders. Secondly, the structure of Pax2 gene and PAX2 protein, as well as the function of Pax2 gene in neural development, was discussed. Finally, a diagram of the PAX2 protein regulatory network was made and a possible molecular mechanism of Pax2 mutations leading to neurodevelopmental disorders from the perspectives of developmental process and protein function was proposed.

Introduction

Transcription factors (TFs) play an important role in the developing and adult mammalian brain. In the early stage of embryonic development, TFs determine the expression and relative abundance of guide molecules, mediating progenitor cell differentiation into different types of nerve cells. In the adult stage, TFs are still involved in neurogenesis. The latest research suggests that TFs are responsible for the production of glutamatergic neurons in the adult dentate gyrus of mice.1 Dysregulation of TFs has been implicated in a number of diseases, including neurodevelopmental disorders2–4 and neurodegenerative diseases.5,6 Using viral tools to deliver certain TFs in the mouse brain to reduce the expression of key genes at the transcription level can rescue neuronal damage.4

Here, we focus on paired box 2 (PAX2), a member of the paired box (PAX) transcription factor family expressed mainly in the kidney and central nervous system. The crucial role of the Pax2 genes in brain development and function have become apparent in recent reports on Pax2-related diseases in a Japanese population describing that the patients with Pax2 mutations exhibit developmental abnormalities, including autism spectrum disorders (ASDs), language delay, or mental retardation.7,8 Emerging evidence demonstrates that PAX2 proteins execute important functions during central nervous system development and in adult neurogenesis.9–15 However, the molecular mechanisms required to establish and maintain these functional connections are not yet fully understood. This review examines the role of Pax2 in brain development and the clinical phenotypes related to neurological aspects in patients with Pax2 mutation, focusing on the expression patterns of PAX2 at different stages. A better understanding of these factors will aid in the development of novel treatments to prevent and/or treat diseases associated with Pax2 mutations.

Pax2-Related Neurodevelopmental Disorders

Developmental biology studies have confirmed that the regulation of transcription levels is involved in the process of neurodevelopment, and TFs has become a key gene category that is recognized to be disrupted in neurodevelopmental disorders in the form of a single gene.2 For example, Pax5 and Pax6 in the same gene family as the Pax2 were reported by exome sequencing research to be two candidate risk genes for ASDs,16–18 which is a neurodevelopmental disorder characterized by continuous social interaction and social communication skills deficits and restricted and repetitive behavior patterns, interests or activities.19

Pax2-related diseases are inherited in an autosomal dominant manner. Patients with Pax2 mutations show renal and eye abnormalities, such as renal dysplasia or optic nerve coloboma,20–22 as well as neurodevelopmental disorders such as ASDs, intellectual disability, epilepsy and developmental delay (Figure 1; Table 1), 7,8,23–30 According to the Leiden Open Variation Database (), the total number of public variants of the Pax2 gene currently reported is 334, including deletions, insertions, substitutions and duplications of cDNA reference sequences, these variations are mostly located in exons 2, 3, 7, and 8. The most frequently occurring mutation described is c.76dup of exon 2 (previously called c.619insG), which introduces a stop codon 27 amino acids downstream from the homoguanine tract. Nevertheless, the molecular mechanism of how these mutations affect protein function and lead to neurodevelopmental disorders is not clear. Understanding the expression and function of the Pax2 gene may give some clues.

Figure 1

Pax2 mutation spectrum (NM_003987.5) and the structure of protein in human. (A) Pax2 mutation spectrum. Mutations identified in patients with neurodevelopment disorders are indicated with red characters, most common mutation identified in patients with renal and eye abnormalities is in blue. (B) PAX2 protein contains a DNA binding domain of 128 amino acids (paired domain, PD, yellow) at the amino-terminal and a transactivation domain (TD, Orange) at the carboxyl-terminal, and a conserved octapeptide (OP, purple) motif and a homeodomain (HD, brown). The exons 1–4 encode the PD, exon 5 encodes the OP, and exons 8–11 encode the TD of the PAX2 protein.

Table 1

List of Pax2 Variants in Neurodevelopmental Disorders

Patient	Sex	Exon	DNA Change (cDNA)	Protein	Phenotype	Reference
1	M	2	c.68delT	p.Leu23fs	Growth retardation	[22]
2	F	2	c.76delG	p.Val26fs	Developmental delay; Intellectual disability; Microcephaly	[24]
3	M	2	c.76dup	p.Val26GlyfsTer28	Autism spectrum disorder	[7]
4	F	2	c.76dup	p.Val26GlyfsTer28	Hydrocephalus resulting from a Chiari malformation type 1	[28]
5	M	2	c.76dup	p.Val26GlyfsTer28	Developmental delay; Delayed myelination	[8]
6	M	2	c.76dup	p.Val26GlyfsTer28	Moderately intellectual disability; Microcephalic	[27]
7	M	2	c.129–150del22	p.Glu43fs	Seizures
8	F	2	c.129–150del22	p.Glu43fs	Seizures; Abnormal EEG	[22]
9	M	2	c.129–150del22	p.Glu43fs	Intellectual disability
11	M	3	c.343C>T	p.Arg115Ter	Growth retardation	[24]
10	M	3	c.392delG	p.Pro130His	Intellectual disability	[23]
12	M	4	c.497–2A>G	splice site	Autism spectrum disorder	[7]
13	M	5	c.561delC	p.Asn188fs	Seizures	[26]
14	F	–	Long arm deletion on chromosome 10	–	Inadequate psychomotor development	[29]

Nuclear Transcription Factor PAX2

Pax was first isolated and identified through its functions in the Drosophila segmentation gene.31 Based on sequence homology with the Drosophila paired box, nine murine genes containing the paired box have since been isolated, termed Pax1 to Pax9, which together constitute the Pax gene family.32 Throughout the evolutionary process, the Pax gene family has remained highly conserved among different organisms, such as humans, mice, zebrafish33 and chickens.34 The encoded nuclear TFs have a similar structure, with a DNA binding domain of 128 amino acids (paired domain, PD) at the amino-terminal and a transactivation domain (TD) at the carboxyl-terminal. The protein contains a conserved octapeptide (OP) motif and a partial or full homeodomain (HD).35 According to the protein structure, the Pax gene family is divided into four subgroups (Pax1 and Pax9, Pax2, Pax5 and Pax8; Pax3 and Pax7; Pax4 and Pax6).36 The Pax2 gene is a member of the second subgroup, located on mouse chromosome 19 and human chromosome 10q24. In humans, Pax2 consists of 12 exons, with a total length of approximately 70 kb, exons 1–4 encode the PD, exon 5 encodes the OP sequence, and exons 8–11 encode the TD of the PAX2 protein (Figure 1). Mutation or deletion of the OP leads to increased transactivation.37 For example, the deletion of cytosine in exon 5 of patient 13 causes increased gene expression and exhibits seizures (Table 1). The insertion of guanine in exon 2 of patients 3–6, which may cause premature termination of transcription and change the DNA binding function of PD,21 resulting in insufficient PAX2 haplotypes, affecting protein function, and may cause patients to exhibit developmental delays and intellectual disability. While Pax2 consists of 10 exons spanning approximately 91.7 kb in mice, the corresponding translated peptide sequences of human and mouse exon 6 are identical. Studies have shown that the mouse Pax2 gene has 63–67% homology with Drosophila and 69–72% homology with the human gene.38

Alternative splicing is an important mechanism by which eukaryotic genes express a variety of complex proteins. Alternative splicing of PAX2 mRNAs lead to four isoforms in human and mice, including PAX2a, PAX2b, PAX2c, and PAX2d.39–41 PAX2a and PAX2b were first identified in mice, and PAX2c was subsequently discovered in human and mouse kidney. Neither PAX2b nor PAX2c contain exon 6. PAX2c contains an added exon 10 containing 83 bp, causing an exon 11 reading frame migration that leads to the early termination of transcription. PAX2d was also found in the human kidney cDNA library. In this isoform, the first 19 bp of exon 12 are deleted, resulting in the stop codon of the PAX2d transcript being 64 bp downstream of the conventional stop codon. This alternative splicing generates a new reading frame and an extended coding region at the carboxyl terminus, which is widely conserved between human and mice.

Pax2 Gene and Neural Development

Some studies on rodents and human embryos have determined the expression pattern of PAX2 protein in the developmental stage, and the results indicate that expression of the PAX2 protein in the spinal cord and brain during human embryonic development and the description of the role of PAX2 in rodent neurogenesis are very homologous. Pax2 is involved in the early development of mouse embryos, it is expressed in the two borders of the neural plate at E7.5,42 then, neural plate folds to form the neural groove and then closes to generate the neural tube.43 PAX2 is expressed during the development of the neural plate into the neural tube, the neural tube is subdivided into the forebrain, midbrain, hindbrain, and spinal cord.44 Subsequently, PAX2 is expressed in the forebrain,45 midbrain-hindbrain boundary,46 hypothalamus, spinal cord,47 and cerebellum,48 The best studied brain areas in relation to Pax2 are the midbrain-hindbrain, spinal cord, and the cerebellum. The specific function of Pax2 in the hypothalamus is still unclear. The gene dosage of Pax2 and Pax5 in the same subgroup is vital for the function of the organizing center at the midbrain-hindbrain boundary.49 The Pax2 mutant phenotype is strongly influenced by the genetic background of the mouse strain in which it is analyzed. In the C3H/He strain, the hindbrain and cerebellum were completely absent, while in the C57BL/6 strain, brain development was almost normal.50,51 In Pax2 and Pax5 double mutant embryos, the midbrain and hindbrain and cerebellum are absent, however, mutations in Pax2 gene alone do not necessarily affect the production of abnormal phenotypes., suggesting that Pax2 and Pax5 compensate for each other.52

During later embryonic stages (maintenance phase), Pax2, Wnt1 and Fgf8 work together to maintain the midbrain-hindbrain boundary function.49 In the midbrain, the Pax2 expression domain is the source of midbrain dopaminergic neurons.53,54 In the spinal cord and cerebellum, the Pax2 gene was found to be closely related to GABAergic neurons.55 GABAergic interneurons derived from PAX2-positive cells expressed in the cerebellar parenchyma at E13.5 subsequently proliferated to produce different types of inhibitory interneurons, including Golgi and Lugaro cells in the granular layer and basket and stellate cells in the molecular layer.56,57 Studies have shown that PAX2 is expressed in newborn GABAergic neurons on the dorsal spinal cord and is considered a key marker of GABAergic neurons.47 PAX2 acts together with other transcription factors, PTF1A, PRDM13 and LBX1, to participate in the differentiation of GABA precursor neurons and promote the fate of GABAergic neurons,58–61 maintaining continuous expression in GABAergic neurons in adult rats.15 A recent study also concluded that PAX2 is an effective inhibitor of the expression of the Purkinje cell marker FOXP2 and the Purkinje cell differentiation program, and its expression seems to induce the differentiation program characteristics of inhibitory interneurons.62 In our recent research, using CRISPR/Cas9 technology, a Pax2 heterozygous gene deletion mouse (Pax2± KO mice) was constructed. The results of autism-related behavioral tests showed that Pax2± KO mice had significantly increased self-grooming behavior.63 Further study to explore the regulatory mechanism of Pax2 gene deletion leading to autism-like behavior will help to understand the pathogenesis of Pax2-related diseases.

Excitation-inhibitory (E-I) imbalance is considered to be a characteristic of various neurodevelopmental disorders. Large-scale exome sequencing results have revealed that risk genes are enriched in the excitatory and inhibitory neuron lineages.64 GABA is a major inhibitory neurotransmitter widely distributed in the brain and plays an important role in maintaining E-I balance.65–68 It has been reported that GABA or GABA receptor levels in multiple brain regions of children or adults with ASDs are significantly reduced.69–72 Exome sequencing has demonstrated that mutations in the gene that encodes the GABAA receptor subunit disrupt GABA transmission in epilepsy and in people with intellectual disabilities.73–76 Pax2 regulates the cell fate of GABAergic precursor neurons during the development of the cerebellum and spinal cord. Mutation or deletion of Pax2 may lead to impaired transcription regulation, altered GABA levels in the central nervous system, impaired synaptic E-I balance, and neural circuit abnormalities resulting in neurodevelopmental disorders (Figure 2).

Figure 2

A possible mechanism by which Pax2 mutations leads to neurodevelopmental disorders. On the one hand, Pax2 gene mutations affect the normal formation of the midbrain and hindbrain boundary leading to neurodevelopmental disorders, such as autism spectrum disorders. On the other hand, Pax2 gene disrupts the excitation-inhibitory (E-I) balance in the brain leading to neurodevelopmental disorders by regulating the cell fate of GABAergic interneurons.

Transcriptional Networks Associated with PAX2

The PAX2 recognizes its target gene through the DNA binding function of the pairing domain. Transcription ability is determined by the C-terminal fragment module, which consists of an activation sequence and an inhibitor sequence.77 Therefore, the PAX2 transcription factor acts as both a transcriptional activator and a repressor. The downstream expansion target genes and binding genes of PAX2 have been gradually discovered (Figure 3). It has been reported that GDNF is the target gene of PAX2 in medulloblastoma.78 The complex of transforming factors EYA1 and PAX2 upregulates Six2 and GDNF, which was identified by cDNA microarray analysis. The five genes restricted by PAX2 in the midbrain-hindbrain boundary are En2, Brn1, Sef, Tapp1 and Ncrms.79 PAX2 activates these genes to control the development of the midbrain and cerebellum. Among them, En2 and Brn1 are risk genes for ASDs;80–82 A recent study revealed that PAX2 induces the expression of cyclin D1 by activating AP-1 and promotes the proliferation of colon cancer cells;83 PAX2 activates WNT4 during the development of mammals expression.84 At the same time, PAX2 is also regulated by upstream transcription factors. PAX2 is the direct transcription target of P53;85 the tumor suppressor MENIN inhibits PAX2 through WT1;86 ASH2L participates in the promotion of endometrial cancer progression by upregulating transcription of PAX2.87 During early neurogenesis, PRDM13 regulates and suppresses PAX2, a neuron-specific factor in the neuronal lineage. PAX2 binding sites have also been continuously discovered, including IL-5, PTIP, EYAL, LIM1, PLAC8.87,88 We speculate that Pax2 mutations may affect some factors in the transcriptional network, resulting in abnormal development of the midbrain and hindbrain leading to neurodevelopmental disorders.

Figure 3

PAX2-associated transcriptional networks and PAX2-binding proteins. As a gene encoded transcription factor, PAX2 is not only regulated by upstream transcription factors, but also can activate or inhibit the expression of downstream genes. +: activate; -: inhibit.

Conclusion

In the nervous system, the PAX2 protein participates in the formation of the mid- hindbrain boundary during development, regulates fate determination of precursor neurons, and retains expression in mature cells. However, the specific molecular mechanism of Pax2 regulation remains unclear. Does Pax2 continue to regulate the fate of inhibitory interneurons in the cerebral cortex and other brain regions after development? Which downstream target genes does Pax2 specifically regulate, and how do they function? These questions can be addressed using the latest single-cell transcriptome sequencing technology. Patients with Pax2 mutations exhibit neurodevelopmental disorders, but it is unclear which neurodevelopmental processes affected. The construction of Pax2 conditional gene knockout mice may help to further clarify the pathogenesis of neurodevelopmental disorders in patients with Pax2 mutations.