Targeting impaired autophagy as a therapeutic strategy for Koolen-de Vries syndrome

ABSTRACT

Koolen-de Vries syndrome (KdVS) is a genomic disorder characterized by intellectual disability, heart failure, hypotonia and congenital malformations, which is caused by haploinsufficiency of KANSL1. Because the pathogenesis of the disease is unknown, there is still no effective treatment. Here, we discuss our recent work identifying KANSL1 as an essential gene for macroautophagy/autophagy. We find that KANSL1 modulates autophagosome-lysosome fusion for cargo degradation by transcriptionally regulating Stx17 expression. Kansl1 heterozygous mice exhibit impaired neuronal and cardiac functions, resulting from the obstruction of autophagic clearance of damaged mitochondria and accumulation of reactive oxygen species in these tissues. Furthermore, we discovered an FDA-approved drug, 13-cis retinoic acid, is capable of alleviating these mitophagic defects and neurobehavioral abnormalities in Kansl1 heterozygous mice by promoting autophagosome-lysosome fusion via directly binding to STX17 and SNAP29. Our study provides the proof of concept to set up a link between KANSL1, autophagic defects and KdVS, and also proposes a therapeutic strategy for treatment of KdVS.

The estimated prevalence of KdVS is approximately 1 in 16,000 individuals. Previous studies reported that haploinsufficiency in the KANSL1 gene causes this disease. However, due to the lack of understanding of its pathogenesis, there is currently no effective therapy for this disease. In our recently published work, we reveal that defects in the selective elimination of mitochondria by autophagy caused by KANSL1 gene deletion is a previously unrecognized pathogenic mechanisms leading to KdVS. Importantly, based on this finding, we proposed a drug repurposing strategy that targets the impaired autophagy for the treatment of this disease [1].

It has been well established that autophagy is a conserved lysosomal degradation process critical for cellular homeostasis. Although many neurodegenerative diseases have been reported to be closely associated with autophagy abnormalities, many open questions and challenges remain to be addressed regarding the physiological and pathological function of autophagy. In our recent study, using a high-content siRNA screen we determined that the protein encoded by the KdVS pathogenic gene, KANSL1, is critical for autophagic activity. As the mutations in the KANSL1 gene found in KdVS patients usually result in its single-copy defect, we constructed Kansl1 heterozygous mice and confirmed that the autophagic activity in the heart and brain in this mouse model is significantly reduced. To further investigate the molecular mechanisms by which KANSL1 deficiency leads to autophagic dysfunction, we performed a combined analysis of RNA-seq and ChIP-seq and discovered that KANSL1 transcriptionally regulates the expression of Stx17, a key gene for autophagosome-lysosome fusion. In KANSL1-deficient cells, Stx17 transcription is significantly downregulated, and the process of autophagosome-lysosome fusion is blocked (Figure 1). Eventually, damaged mitochondria cannot be properly cleared particularly in those mitochondria-rich tissues such as heart and brain, resulting in accumulated reactive oxygen species, which in turn triggers a destruction in cardiac function and learning and memory ability in Kansl1 heterozygous mice.

Figure 1.

Proposed model for the mechanisms of KANSL1 in a Koolen-de Vries syndrome mouse model through autophagy. KANSL1 modulates Stx17 transcription in neurons. Sufficient STX17 ensures the regular progress of the autophagosome-lysosome fusion process, which is essential for the clearance of damaged mitochondria and the healthy states of neurons (left). However, in the Koolen-de Vries syndrome mouse model, KANSL1 deficiency disrupts Stx17 transcription and impairs autophagosome-lysosome fusion process, leading to the accumulation of damaged mitochondria in neurons and the behavioral abnormality in the KdVS mouse (right). When treated with 13-cis retinoic acid, the KdVS mouse exhibits phenotypes similar to the wild type.

We next ask whether an intervention of the dysfunction in autophagosome-lysosome fusion can alleviate the KdVS-like disorders observed in Kansl1 heterozygous mice. To this end, we screened a natural small molecule compound library in KANSL1-deficient neurons using the mitophagy indicator mt-Keima. Fortunately, we demonstrated that 13-cis retinoic acid (13-cis RA), a drug previously approved by the FDA for the treatment of nodulocystic acne, is able to restore the mitochondrial autophagic activity in KANSL1-deficient neurons (Figure 1). Subsequent in vitro and in vivo studies revealed that 13-cis RA directly binds to the STX17 protein and enhances its interaction with the other autophagic fusion proteins, thereby promoting the fusion process of autophagy. In mice, 13-cis RA effectively relieves mitochondrial autophagy defects in neurons, which increases the dendritic spine density and improves the behavioral abnormality in KdVS mouse models.

Given that 13-cis RA exhibits an impressive effect in treating KdVS mice, it is worthy of further verification through clinical trials. It should be noted that this drug has been found to have serious teratogenic side effects when treating nodulocystic acne in the clinic. Although according to the dose conversion between human and mouse, the dose of 13-cis RA used in KdVS mice is lower than that of its clinical usage for human, the drug safety issues should not be neglected in clinical trial of 13-cis RA-based therapy of KdVS in the future. In addition, as one of the retinoid family members, 13-cis RA has some isomers and related-precursors, such as all-trans RA, 9-cis RA and vitamin A, some of which have a better safety profile. Therefore, it would also be of value to investigate the effects of these other compounds on autophagy in KANSL1-deficient cells as well as KdVS mouse models and patients. These further studies may provide safer and more effective means of treating KdVS.

Another intriguing question worth noting is that 13-cis RA may also have therapeutic prospects for a class of diseases caused by downstream blockade of autophagy. Nowadays, although many upstream autophagy agonists have been discovered, very few agonists have been identified to directly target downstream autophagy, especially in the fusion stage of autophagosomes. Therefore, the treatment of such diseases still faces enormous challenges. For instance, some genomic diseases, such as Kufor-Rakeb syndrome and Danon disease, are caused by mutations in autophagosome fusion-related genes. In addition, certain chemicals, such as fluoride, are capable of inducing developmental neurotoxicity, which is also thought to be caused by the obstruction of the autophagosome-lysosome fusion process. Also of note is that several recent studies have reported that ORF3a of the COVID-19 virus SARS-CoV-2 inhibits autophagy activity by blocking fusion of autophagosomes or amphisomes with lysosomes, which may contribute to the infectivity and pathogenicity of SARS-CoV-2. All of the above diseases are closely related to the abnormal fusion process of autophagy. Therefore, further studies will be required to determine whether 13-cis RA has therapeutic prospects in these diseases. To summarize, our study not only reveals that mitochondrial autophagy dysfunction is a key pathogenesis of KdVS, but also proposes that 13-cis RA could be a potential therapeutic agent for diseases caused by downstream blockade of autophagy such as KdVS.

Funding Statement

This work was supported by the National Natural Science Foundation of China [No. 82025028, No. 31871390 and No. 81522034]; National Key Research and Development Program of China [2021YFA1300200].

Disclosure statement

No potential conflict of interest was reported by the author(s).