Kansl1 haploinsufficiency impairs autophagosome-lysosome fusion and links autophagic dysfunction with Koolen-de Vries syndrome in mice

A recent study shows a link between autophagy and a rare genetic disorder, Koolen-de Vries syndrome, caused by loss of KANSL1. An FDA-approved drug, 13-cis retinoic acid, was able to rescue autophagy in the absence of KANSL1.

Dysregulation of autophagy can cause systemic diseases

Autophagy is a complex cellular process that plays a critical role in not only cellular homeostasis, but also tissue homeostasis. This pathway degrades macromolecules and damaged organelles at times of stress and starvation to enable recycling of basic biomolecules. Disruption of autophagy can lead to a variety of diseases including cancer and heart, liver, and neurological diseases, but the precise etiology is typically unknown.¹ A better understanding of the role that autophagy plays in neuronal diseases can lead to better therapies for these diseases.

Koolen-de Vries syndrome (KdVS) is a rare genetic disorder caused by deletion, truncation, or haploinsufficiency of KANSL1 (KAT8 regulatory NSL complex subunit 1). KdVS is characterized by developmental delay, intellectual disability, cardiac and renal defects, epilepsy and seizures, and hypotonia.² The pathogenesis of the disease is unknown and there are currently no effective treatments available.

The functions of KANSL1

KANSL1 plays a key role in mitochondrial function

KANSL1 encodes a nuclear protein that participates in chromatin modification. KANSL1 along with seven other proteins forms the NSL (non-specific lethal) complex. The NSL complex plays an important role in histone acetylation, which regulates gene transcription. KANSL1 is a key scaffolding protein for the NSL complex and is required for its catalytic activity. KANSL1 also localizes to microtubules to regulate mitosis and to mitochondria to regulate mitochondrial respiration and mtDNA transcription. The complete role of KANSL1 in mitochondrial function is still unknown.³

A recent study by Li et al. pieced together a puzzle linking KANSL1 to autophagy and neuronal and cardiac function.⁴

KANSL1 is a regulator of autophagosome-lysosome function

The authors used siRNA screens and assays for autophagic activity to identify autophagy regulators. KANSL1 was the only candidate that showed reduced autophagic degradation in the initial screen. To examine whether these observations also occurred in primary cells, mouse embryonic fibroblasts (MEFs) from tamoxifen-inducible knockout (KANSL1fl/fl/CAG-cre) embryos were examined. These KANSL1-deficient MEFs had enhanced autophagosome formation, high levels of p62 (which recruits cargo to autophagic membranes), and increased accumulation of autophagosomes after starvation.

This autophagic impairment was narrowed down to either increased autophagosome formation or reduced degradation of autophagosomes. KANSL1 was not found to play a role in autophagosome formation, since KANSL1-deficient cells did not have further increased ratios of LC3B-11/LC3B-I after treatment with an autophagic inhibitor. A tandem dye system, in which autophagosomes appear yellow due to a combination of red and green staining and autolysosomes appeared red due to loss of green staining upon acidification, was used to determine that KANSL1-deficient cells had reduced levels of autophagosome degradation. KANSL1-deficient cells did not have altered lysosome abundance or function but did lack colocalization of autophagosomes and lysosomes. This data suggests that KANSL1 affects autophagosome-lysosome fusion.

Transcriptional control of autophagy

To determine whether this disruption in autophagy was due to transcriptional control of autophagy genes by KANSL1, RNA-seq data was cross-referenced with CHIP-seq data to identify a subset of vesicle and autophagy genes. Ten genes were identified as autophagy-related genes, only three of which were known to be involved in autophagosome-lysosome fusion: STX17, PIK3R4, and ATG14. STX17 was the most significantly decreased in KANSL1-deficient cells indicating it might be a key target of KANSL1. Further studies showed KANSL1 was able to bind to the STX17 promoter and drive STX17 transcription. Additionally, autophagy disruption in KANSL1-deficient cells was rescued both by cleavage-resistant KANSL1 and STX17, indicating that KANSL1 regulates autophagy through STX17.

Koolen-deVries syndrome and KANSL1

In an attempt to generate whole-body knockout mice by crossing KANSL1^fl/fl mice with EIIA-cre, the researchers found that complete knockout of KANSL1 in mice is embryonic lethal. Interestingly, KANSL1^+/- MEFs had similar autophagic defects to KANSL1^-/- MEFs, and KANSL1 haploinsufficiency is sufficient to alter STX17 expression. These impairments in autophagy and STX17 expression extended to cardiac tissue and the hippocampus. As seen in KdVS, KANSL1^+/- mice had decreased object recognition and spatial learning compared to KANSL1^+/+ mice. Even though the KANSL1^+/- mice had no neuronal loss, they did have decreased dendritic spine density, likely contributing to hippocampal dysfunction.

Mitochondrial autophagy, also known as mitophagy, plays an important role in the maintenance of neurons and neuronal function. This along with the ability of KANSL1 to localize to mitochondria led the investigators to examine whether KANSL1 regulates mitophagy. Hippocampal cells from KANSL1^+/- mice had decreased levels of mitophagy compared to wild type controls. This decrease in mitophagy was partially rescued by STX17 overexpression.

Consistent with the previous findings, damaged mitochondria accumulated in KANSL1^+/- neurons due to the defect in mitophagy. These defects resulted in increased reactive oxygen species inside primary neurons and may be the cause of the hippocampal and cardiac phenotypes observed in KANSL1-deficient mice.

Therapeutic development to treat Koolen-deVries syndrome

Since currently there are no treatments available for KdVS, a small molecule screen was used to identify targets that rescued mitophagy in the KANSL1-deficient cells. 13-cis retinoic acid (13-cis RA) was the most effective candidate at rescuing mitophagy. 13-cis RA decreased reactive oxygen species and improved colocalization of autophagosomes and lysosomes inside primary neurons. This molecule increased the interaction among the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins STX17, VAMP8, YKT6, STX7, and SNAP29 without increasing the levels of these proteins. SNARE proteins localize to membranes and during autophagy create a bridge between the autophagsome and lysosome tethering the membranes together facilitating fusion.⁵ These enhanced SNARE interactions upon 13-cis RA treatment promote autophagosome-lysosome fusion pushing autophagy towards completion. Additionally, 13-cis RA treatment rescued the hippocampal impairments of spatial learning and memory in KANSL1^+/- mice.

The identification of KANSL1 as a key factor regulating STX7 expression and autophagosome-lysosome fusion has not only provided mechanistic insight into the pathology of KdVS, but also has provided novel therapeutic options for this rare disease.

Name	Role in Autophagy	Catalog #	Applications	Reactivity	Host	Clonality
STX7	Autophagosome-lysosome fusion.	A304-512A	IP, WB	Hu, Ms	Rabbit	Polyclonal
MOF/MYST1	Not involved in autophagy, but a key component of the NSL complex	A300-994A	WB	Hu, Ms	Rabbit	Polyclonal

References:

Wirawan E, Berghe T vanden, Lippens S, Agostinis P, Vandenabeele P. Autophagy: for better or for worse. Cell Res. 2012;22(1):43-61. doi:10.1038/cr.2011.152
Ciaccio C, Dordoni C, Ritelli M, Colombi M. Koolen-de Vries Syndrome: Clinical Report of an Adult and Literature Review. Cytogenet Genome Res. 2016;150(1):40-45. doi:10.1159/000452724
Sheikh BN, Guhathakurta S, Akhtar A. The non‐specific lethal ( <scp>NSL</scp> ) complex at the crossroads of transcriptional control and cellular homeostasis. EMBO Rep. 2019;20(7). doi:10.15252/embr.201847630
Li T, Lu D, Yao C, et al. Kansl1 haploinsufficiency impairs autophagosome-lysosome fusion and links autophagic dysfunction with Koolen-de Vries syndrome in mice. Nat Commun. 2022;13(1):931. doi:10.1038/s41467-022-28613-0
Wang Y, Li L, Hou C, et al. SNARE-mediated membrane fusion in autophagy. Semin Cell Dev Biol. 2016;60:97-104. doi:10.1016/j.semcdb.2016.07.009