Ataxia with giant axonopathy in Acbd5-deficient mice halted by adeno-associated virus gene therapy.

Acyl-CoA binding domain containing 5 (ACBD5) is a critical player in handling very long chain fatty acids (VLCFA) en route for peroxisomal ß-oxidation. Mutations in ACBD5 lead to the accumulation of VLCFA and patients present retinal dystrophy, ataxia, psychomotor delay and a severe leukodystrophy. Using CRISPR/Cas9, we generated and characterized an Acbd5 Gly357* mutant allele. Gly357* mutant mice recapitulated key features of the human disorder, including reduced survival, impaired locomotion and reflexes, loss of photoreceptors, and demyelination. The ataxic presentation of Gly357* mice involved the loss of cerebellar Purkinje cells and a giant axonopathy throughout the CNS. Lipidomic studies provided evidence for the extensive lipid dysregulation caused by VLCFA accumulation. Following a proteomic survey, functional studies in neurons treated with VLCFA unravelled a deregulated cytoskeleton with reduced actin dynamics and increased neuronal filopodia. We also show that an adeno-associated virus-mediated gene delivery ameliorated the gait phenotypes and the giant axonopathy, also improving myelination and astrocyte reactivity. Collectively, we established a mouse model with significance for VLCFA-related disorders. The development of relevant neuropathological outcomes enabled the understanding of mechanisms modulated by VLCFA and the evaluation of the efficacy of preclinical therapeutic interventions.

Plasmalogens regulate the AKT-ULK1 signaling pathway to control the position of the axon initial segment.

The axon initial segment (AIS) is a specialized region in neurons that encompasses two essential functions, the generation of action potentials and the regulation of the axodendritic polarity. The mechanism controlling the position of the axon initial segment to allow plasticity and regulation of neuron excitability is unclear. Here we demonstrate that plasmalogens, the most abundant ether-phospholipid, are essential for the homeostatic positioning of the AIS. Plasmalogen deficiency is a hallmark of Rhizomelic Chondrodysplasia Punctata (RCDP) and Zellweger spectrum disorders, but Alzheimer's and Parkinson's disease, are also characterized by plasmalogen defects. Neurons lacking plasmalogens displaced the AIS to more distal positions and were characterized by reduced excitability. Treatment with a short-chain alkyl glycerol was able to rescue AIS positioning. Plasmalogen deficiency impaired AKT activation, and we show that inhibition of AKT phosphorylation at Ser473 and Thr308 is sufficient to induce a distal relocation of the AIS. Pathway analysis revealed that downstream of AKT, overtly active ULK1 mediates AIS repositioning. Rescuing the impaired AKT signaling pathway was able to normalize AIS position independently of the biochemical defect. These results unveil a previously unknown mechanism that couples the phospholipid composition of the neuronal membrane to the positional assembly of the AIS.

Profilin 1 delivery tunes cytoskeletal dynamics toward CNS axon regeneration.

After trauma, regeneration of adult CNS axons is abortive, causing devastating neurologic deficits. Despite progress in rehabilitative care, there is no effective treatment that stimulates axonal growth following injury. Using models with different regenerative capacities, followed by gain- and loss-of-function analysis, we identified profilin 1 (Pfn1) as a coordinator of actin and microtubules (MTs), powering axonal growth and regeneration. In growth cones, Pfn1 increased actin retrograde flow, MT growth speed, and invasion of filopodia by MTs, orchestrating cytoskeletal dynamics toward axonal growth. In vitro, active Pfn1 promoted MT growth in a formin-dependent manner, whereas localization of MTs to growth cone filopodia was facilitated by direct MT binding and interaction with formins. In vivo, Pfn1 ablation limited regeneration of growth-competent axons after sciatic nerve and spinal cord injury. Adeno-associated viral (AAV) delivery of constitutively active Pfn1 to rodents promoted axonal regeneration, neuromuscular junction maturation, and functional recovery of injured sciatic nerves, and increased the ability of regenerating axons to penetrate the inhibitory spinal cord glial scar. Thus, we identify Pfn1 as an important regulator of axonal regeneration and suggest that AAV-mediated delivery of constitutively active Pfn1, together with the identification of modulators of Pfn1 activity, should be considered to treat the injured nervous system.

Leukodystrophy caused by plasmalogen deficiency rescued by glyceryl 1-myristyl ether treatment.

Plasmalogens are the most abundant form of ether phospholipids in myelin and their deficiency causes Rhizomelic Chondrodysplasia Punctata (RCDP), a severe developmental disorder. Using the Gnpat-knockout (KO) mouse as a model of RCDP, we determined the consequences of a plasmalogen deficiency during myelination and myelin homeostasis in the central nervous system (CNS). We unraveled that the lack of plasmalogens causes a generalized hypomyelination in several CNS regions including the optic nerve, corpus callosum and spinal cord. The defect in myelin content evolved to a progressive demyelination concomitant with generalized astrocytosis and white matter-selective microgliosis. Oligodendrocyte precursor cells (OPC) and mature oligodendrocytes were abundant in the CNS of Gnpat KO mice during the active period of demyelination. Axonal loss was minimal in plasmalogen-deficient mice, although axonal damage was observed in spinal cords from aged Gnpat KO mice. Characterization of the plasmalogen-deficient myelin identified myelin basic protein and septin 7 as early markers of dysmyelination, whereas myelin-associated glycoprotein was associated with the active demyelination phase. Using in vitro myelination assays, we unraveled that the intrinsic capacity of oligodendrocytes to ensheath and initiate membrane wrapping requires plasmalogens. The defect in plasmalogens was rescued with glyceryl 1-myristyl ether [1-O-tetradecyl glycerol (1-O-TDG)], a novel alternative precursor in the plasmalogen biosynthesis pathway. 1-O-TDG treatment rescued myelination in plasmalogen-deficient oligodendrocytes and in mutant mice. Our results demonstrate the importance of plasmalogens for oligodendrocyte function and myelin assembly, and identified a novel strategy to promote myelination in nervous tissue.

Peripheral nervous system plasmalogens regulate Schwann cell differentiation and myelination.

Rhizomelic chondrodysplasia punctata (RCDP) is a developmental disorder characterized by hypotonia, cataracts, abnormal ossification, impaired motor development, and intellectual disability. The underlying etiology of RCDP is a deficiency in the biosynthesis of ether phospholipids, of which plasmalogens are the most abundant form in nervous tissue and myelin; however, the role of plasmalogens in the peripheral nervous system is poorly defined. Here, we used mouse models of RCDP and analyzed the consequence of plasmalogen deficiency in peripheral nerves. We determined that plasmalogens are crucial for Schwann cell development and differentiation and that plasmalogen defects impaired radial sorting, myelination, and myelin structure. Plasmalogen insufficiency resulted in defective protein kinase B (AKT) phosphorylation and subsequent signaling, causing overt activation of glycogen synthase kinase 3ß (GSK3ß) in nerves of mutant mice. Treatment with GSK3ß inhibitors, lithium, or 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) restored Schwann cell defects, effectively bypassing plasmalogen deficiency. Our results demonstrate the requirement of plasmalogens for the correct and timely differentiation of Schwann cells and for the process of myelination. In addition, these studies identify a mechanism by which the lack of a membrane phospholipid causes neuropathology, implicating plasmalogens as regulators of membrane and cell signaling.

Early axonal loss accompanied by impaired endocytosis, abnormal axonal transport, and decreased microtubule stability occur in the model of Krabbe's disease.

In Krabbe's disease (KD), a leukodystrophy caused by ß-galactosylceramidase deficiency, demyelination and a myelin-independent axonopathy contributes to the severe neuropathology. Beyond axonopathy, we show that in Twitcher mice, a model of KD, a decreased number of axons both in the PNS and in the CNS, and of neurons in dorsal root ganglia (DRG), occurred before the onset of demyelination. Despite the early axonal loss, and although in vitro Twitcher neurites degenerated over time, Twitcher DRG neurons displayed an initial neurite overgrowth and, following sciatic nerve injury, Twitcher axons were regeneration-competent, at a time point where axonopathy was already ongoing. Psychosine, the toxic substrate that accumulates in KD, induced lipid raft clustering. At the mechanistic level, TrkA recruitment to lipid rafts was dysregulated in Twitcher neurons, and defective activation of the ERK1/2 and AKT pathways was identified. Besides defective recruitment of signaling molecules to lipid rafts, the early steps of endocytosis and the transport of endocytic and synaptic vesicles were impaired in Twitcher DRG neurons. Defects in axonal transport, specifically in the retrograde component, correlated with decreased levels of dynein, abnormal levels of post-translational tubulin modifications and decreased microtubule stability. The identification of the axonal defects that precede demyelination in KD, together with the finding that Twitcher axons are regeneration-competent when axonopathy is already installed, opens new windows of action to effectively correct the neuropathology that characterizes this disorder.

The importance of ether-phospholipids: a view from the perspective of mouse models.

Ether-phospholipids represent an important group of phospholipids characterized by an alkyl or an alkenyl bond at the sn-1 position of the glycerol backbone. Plasmalogens are the most abundant form of alkenyl-glycerophospholipids, and their synthesis requires functional peroxisomes. Defects in the biosynthesis of plasmalogens are the biochemical hallmark of the human peroxisomal disorder Rhizomelic Chondrodysplasia Punctata (RCDP), which is characterized by defects in eye, bone and nervous tissue. The generation and characterization of mouse models with defects in plasmalogen levels have significantly advanced our understanding of the role and importance of plasmalogens as well as pathogenetic mechanisms underlying RCDP. A review of the current mouse models and the description of the combined knowledge gathered from the histopathological and biochemical studies is presented and discussed. Further characterization of the role and functions of plasmalogens will contribute to the elucidation of disease pathogenesis in peroxisomal and non-peroxisomal disorders. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.

Alkyl-glycerol rescues plasmalogen levels and pathology of ether-phospholipid deficient mice.

A deficiency of plasmalogens, caused by impaired peroxisomal metabolism affects normal development and multiple organs in adulthood. Treatment options aimed at restoring plasmalogen levels may be relevant for the therapy of peroxisomal and non-peroxisomal disorders. In this study we determined the in vivo efficacy of an alkyl glycerol (AG), namely, 1-O-octadecyl-rac-glycerol, as a therapeutic agent for defects in plasmalogen synthesis. To achieve this, Pex7 knockout mice, a mouse model for Rhizomelic Chondrodysplasia Punctata type 1 characterized by the absence of plasmalogens, and WT mice were fed a control diet or a diet containing 2% alkyl-glycerol. Plasmalogen levels were measured in target organs and the biochemical data were correlated with the histological analysis of affected organs. Plasmalogen levels in all peripheral tissues of Pex7 KO mice fed the AG diet for 2 months normalized to the levels of AG fed WT mice. In nervous tissues of Pex7 KO mice fed the AG-diet, plasmalogen levels were significantly increased compared to control fed KO mice. Histological analysis of target organs revealed that the AG-diet was able to stop the progression of the pathology in testis, adipose tissue and the Harderian gland. Interestingly, the latter tissues are characterized by the presence of lipid droplets which were absent or reduced in size and number when ether-phospholipids are lacking, but which can be restored with the AAG treatment. Furthermore, nerve conduction in peripheral nerves was improved. When given prior to the occurrence of major pathological changes, the AG-diet prevented or ameliorated the pathology observed in Pex7 KO mice depending on the degree of plasmalogen restoration. This study provides evidence of the beneficial effects of treating a plasmalogen deficiency with alkyl-glycerol.

Pleiotropic effects of fenretinide in neuroblastoma cell lines and multicellular tumor spheroids.

The efficacy and mechanism of action of fenretinide (4-HPR), a vitamin A analogue, was investigated in a panel of six neuroblastoma cell lines and multicellular tumor spheroids. The latter are three dimensional cell aggregates and as such, a model for micrometastases. In all cell lines, the production of reactive oxygen species (ROS) increased with 163-680% after 1 h of treatment with 4-HPR. In addition, a decrease of the mitochondrial membrane potential of 30-75% was observed after 4 h of incubation with 4-HPR. A 6-12-fold difference was observed between the IC50 values for cell proliferation and viability between the most sensitive (IMR32) and most resistant (NASS) cell line towards 4-HPR. Flow cytometric analysis showed an increased amount of apoptotic bodies and no cell-cycle arrest. The antioxidant Trolox completely inhibited the accumulation of 4HPR-induced ROS and prevented the 4HPR-associated cytotoxicity. In all neuroblastoma spheroids, 4-HPR induced a complete cytostasis at clinical relevant concentrations (3-10 microM). Immunohistochemical analysis of 4-HPR-treated spheroids showed a decreased staining for proliferation marker Ki-67 and an increased staining for cleaved-PARP, a marker of apoptosis. Our results suggest that 4-HPR might be a promising agent for the treatment of micrometastases and high-risk neuroblastoma.