Neurodegenerative and neurodevelopmental roles for bulk lipid transporters VPS13A and BLTP2

Background: Bridge‐like lipid transfer proteins (BLTPs) mediate bulk lipid transport at membrane contact sites. Mutations in BLTPs are linked to both early‐onset neurodevelopmental and later‐onset neurodegenerative diseases, including movement disorders. The tissue specificity and temporal requirements of BLTPs in disease pathogenesis remain poorly understood.

Objective: The objective of this study was to determine tissue‐specific and aging‐dependent roles for VPS13A and BLTP2 using Drosophila models.

Methods: We generated tissue‐specific knockdowns of the VPS13A ortholog (Vps13) and the BLTP2 ortholog (hobbit) in neurons and muscles of Drosophila. We analyzed age‐dependent locomotor behavior, neurodegeneration, and synapse development and function.

Results: Neuron‐specific loss of the VPS13A ortholog caused neurodegeneration followed by aging‐dependent movement deficits and reduced lifespan, whereas muscle‐specific loss affected only lifespan. In contrast, neuronal loss of the BLTP2 ortholog resulted in severe early‐onset locomotor defects without neurodegeneration, whereas muscle loss impaired synaptogenesis and neurotransmission at the neuromuscular junction.

Conclusions: VPS13A maintains neuronal survival, whereas BLTP2 orchestrates synaptic development. The phenotypic specificity of BLTP function provides mechanistic insights into distinct disease trajectories for BLTP‐associated disorders. 

Methods
Adult climbing assays

For adult climbing, groups of n~10 flies were collected at 1-2 days old and allowed to age for the appropriate time. Then, each group of flies of the appropriate age and genotype was placed into a 50 mL graduated cylinder and allowed to acclimate for 3 min. All flies were then tapped to the bottom of the cylinder, and the number of flies climbing 8 cm in 12 s was counted. This procedure was repeated three times for each group of flies, and the average of the three trials is reported on the graphs. Three independent biological replicates with approximately equal numbers of male and female flies were analyzed for each genotype. Note that the same elav>luc, elav>Vps13(i), 24B>luc, and 24B>Vps13(i) flies were used for climbing assays during aging and for lifespan analysis. During these experiments, flies were transferred to fresh food several times weekly, and the number of living flies was recorded prior to obtaining climbing data weekly.

Histology and neurodegeneration index (NI) quantification

Fly heads were severed and fixed overnight at 4oC in a 6:3:1 solution of ethanol:chloroform:glacial acetic acid, then washed and stored in 70% ethanol for histology. Heads were processed into paraffin using standard histological procedures; serial 5 μm sections spanning the whole fly head were obtained and stained with hematoxylin and eosin (H&E) (UW-Madison Translational Research Initiatives in Pathology lab (TRIP)). Images were obtained using a BioTek Lionheart FX Automated Microscope (10x objective) with Gen5 v.3.11 software, then rotated and cropped post-acquisition in Adobe Photoshop CS6. NI were quantified using the criteria described in Loewen & Ganetzky, 2018. The number and location of vacuoles in the brain determined the NI: 0 = no vacuoles; 1 = a few, small vacuoles mainly in optic lobes in only a few sections; 2 = many vacuoles in many sections, mainly in optic lobes, but possibly some in central brain; 3 = vacuoles become prominent in central brain and are abundant in optic lobes; 4 = many vacuoles in central brain, and some vacuoles in optic lobes and central brain are large. NI scoring was performed blind to age and genotype.

Loewen CA, Ganetzky B. Mito-nuclear interactions affecting lifespan and neurodegeneration in a drosophila model of leigh syndrome. Genetics. 2018 Apr 1;208(4):1535–52.

Body size and lethal phase analysis

Pupa imaging and body size and lethal phase analysis were performed as previously described (Neuman & Bashirullah, 2018).

Neuman SD, Bashirullah A. Hobbit regulates intracellular trafficking to drive insulin-dependent growth during Drosophila development. Development. 2018;145(11):dev161356.