Delineating the phenotype of PNPLA8-related mitochondriopathies.

Pathogenic variants in PNPLA8 have been described either with congenital onset displaying congenital microcephaly, early onset epileptic encephalopathy and early lethality or childhood neurodegeneration with progressive microcephaly. Moreover, a phenotype comprising adulthood onset cerebellar ataxia and peripheral neuropathy was also reported. To our knowledge, only six patients with biallelic variants in PNPLA8 have been reported so far. Here, we report the clinical and molecular characterizations of three additional patients in whom exome sequencing identified a loss of function variant (c.1231C>T, p.Arg411Ter) in Family I and a missense variant (c.1559T>A, p.Val520Asp) in Family II in PNPLA8. Patient 1 presented with the congenital form of the disease while Patients 2 and 3 showed progressive microcephaly, infantile onset seizures, progressive cortical atrophy, white matter loss, bilateral degeneration of basal ganglia, and cystic encephalomalacia. Therefore, our results add the infantile onset as a new distinct phenotype of the disease and suggest that the site of the variant rather than its type is strongly correlated with the disease onset. In addition, these conditions demonstrate some overlapping features representing a spectrum with clinical features always aligning with different age of onset.

Key regulator PNPLA8 drives phospholipid reprogramming induced proliferation and migration in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and leads to the poorest patient outcomes despite surgery and chemotherapy treatment. Exploring new molecular mechanisms of TNBC that could lead to the development of novel molecular targets are critically important for improving therapeutic options for treating TNBC. METHODS: We sought to identify novel therapeutic targets in TNBC by combining genomic and functional studies with lipidomic analysis, which included mechanistic studies to elucidate the pathways that tie lipid profile to critical cancer cell properties. Our studies were performed in a large panel of human breast cancer cell lines and patient samples. RESULTS: Comprehensive lipid profiling revealed that phospholipid metabolism is reprogrammed in TNBC cells. We discovered that patatin-like phospholipase domain-containing lipase 8 (PNPLA8) is overexpressed in TNBC cell lines and tissues from breast cancer patients. Silencing of PNPLA8 disrupted phospholipid metabolic reprogramming in TNBC, particularly affecting the levels of phosphatidylglycerol (PG), phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and glycerophosphocholine (GPC). We showed that PNPLA8 is essential in regulating cell viability, migration and antioxidation in TNBC cells and promoted arachidonic acid and eicosanoid production, which in turn activated PI3K/Akt/Gsk3ß and MAPK signaling. CONCLUSIONS: Our study highlights PNPLA8 as key regulator of phospholipid metabolic reprogramming and malignant phenotypes in TNBC, which could be further developed as a novel molecular treatment target.

Antioxidant Role and Cardiolipin Remodeling by Redox-Activated Mitochondrial Ca2+-Independent Phospholipase A2γ in the Brain.

Mitochondrial Ca2+-independent phospholipase A2γ (iPLA2γ/PNPLA8) was previously shown to be directly activated by H2O2 and release free fatty acids (FAs) for FA-dependent H+ transport mediated by the adenine nucleotide translocase (ANT) or uncoupling protein 2 (UCP2). The resulting mild mitochondrial uncoupling and consequent partial attenuation of mitochondrial superoxide production lead to an antioxidant effect. However, the antioxidant role of iPLA2γ in the brain is not completely understood. Here, using wild-type and iPLA2γ-KO mice, we demonstrate the ability of tert-butylhydroperoxide (TBHP) to activate iPLA2γ in isolated brain mitochondria, with consequent liberation of FAs and lysophospholipids. The liberated FA caused an increase in respiratory rate, which was fully inhibited by carboxyatractyloside (CATR), a specific inhibitor of ANT. Employing detailed lipidomic analysis, we also demonstrate a typical cleavage pattern for TBHP-activated iPLA2γ, reflecting cleavage of glycerophospholipids from both sn-1 and sn-2 positions releasing saturated FAs, monoenoic FAs, and predominant polyunsaturated FAs. The acute antioxidant role of iPLA2γ-released FAs is supported by monitoring both intramitochondrial superoxide and extramitochondrial H2O2 release. We also show that iPLA2γ-KO mice were more sensitive to stimulation by pro-inflammatory lipopolysaccharide, as reflected by the concomitant increase in protein carbonyls in the brain and pro-inflammatory IL-6 release in the serum. These data support the antioxidant and anti-inflammatory role of iPLA2γ in vivo. Our data also reveal a substantial decrease of several high molecular weight cardiolipin (CL) species and accumulation of low molecular weight CL species in brain mitochondria of iPLA2γ-KO mice. Collectively, our results support a key role of iPLA2γ in the remodeling of lower molecular weight immature cardiolipins with predominantly saturated acyl chains to high molecular weight mature cardiolipins with highly unsaturated PUFA acyl chains, typical for the brain.

Homozygous Missense Variation in PNPLA8 Causes Prenatal-Onset Severe Neurodegeneration.

The patatin-like protein family plays an important role in various biological functions including lipid homeostasis, cellular growth, and signaling. Conserved across species, the patatin domain is shared by all 9 members of the PNPLA family without redundancy in the coding sequences. The defective function of PNPLA2, PNPLA6, and PNPLA9 are known to cause mitochondrial-related neurodegeneration. Recently, PNPLA8 has been associated with mitochondrial myopathy and poor weight gain with lactic acidosis in 3 unrelated families. Using whole-exome sequencing, we identified a homozygous novel missense variation c.1874A>G in the patatin domain of PNPLA8. The patient had prenatal-onset severe and progressive neurodegeneration with mortality in infancy.

Loss of function variants in human PNPLA8 encoding calcium-independent phospholipase A2 γ recapitulate the mitochondriopathy of the homologous null mouse.

Mitochondriopathies are a group of clinically heterogeneous genetic diseases caused by defects in mitochondrial metabolism, bioenergetic efficiency, and/or signaling functions. The large majority of proteins involved in mitochondrial function are encoded by nuclear genes, with many yet to be associated with human disease. We performed exome sequencing on a young girl with a suspected mitochondrial myopathy that manifested as progressive muscle weakness, hypotonia, seizures, poor weight gain, and lactic acidosis. She was compound heterozygous for two frameshift mutations, p.Asn112HisfsX29 and p.Leu659AlafsX4, in the PNPLA8 gene, which encodes mitochondrial calcium-independent phospholipase A2 γ (iPLA2 γ). Western blot analysis of affected muscle displayed the absence of PNPLA8 protein. iPLA2 s are critical mediators of a variety of cellular processes including growth, metabolism, and lipid second messenger generation, exerting their functions through catalyzing the cleavage of the acyl groups in glycerophospholipids. The clinical presentation, muscle histology and the mitochondrial ultrastructural abnormalities of this proband are highly reminiscent of Pnpla8 null mice. Although other iPLA2 -related diseases have been identified, namely, infantile neuroaxonal dystrophy and neutral lipid storage disease with myopathy, this is the first report of PNPLA8-related disease in a human. We suggest PNPLA8 join the increasing list of human genes involved in lipid metabolism associated with neuromuscular diseases due to mitochondrial dysfunction.