Npc1a¹ mutants display lethality early in larval development attributable to loss of cholesterol-dependent ecdysone steroid hormone production.

Npc1a¹ mutants fed a diet supplemented with 7-dehydrocholesterol (0.14 mg/g) develop through larval stages, pupate, and eclose as anatomically normal adult animals. Nearly all of these mutants display noraml motor coordination, locomotion, and movement behaviors immediately after eclosion. However, with age, mutants develop obvious locomotory defects, evidenced first as a loss of motor coordination and then as progressive defects in movement behaviors. The onset and progression of these defects varies markedly between individuals. Within 48 hours after eclosion, over 50% of mutant animals appear sluggish, with reduced movement and limited attempts at climbing or evading tactile contact. Within 72 hours, most of these animals show a progressive loss of movement abilities, until movement ceases entirely and the animals die. A subset of mutant animals remain normal and active throughout an 8 day period of observation and only later die a premature, early-onset death. Npc1a¹ mutants maintained in larger populations appear to survive better than isolated individuals, but nevertheless display the same progressive loss of movement behavior and early-onset lethality. Npc1a¹ mutants display a precipitous and continual lethality starting soon after eclosion, with 50% of the population dead at 28 days.

Npc1a¹ mutant brains show an obvious, age-progressive accumulation of filipin (that binds cholesterol aggregates) positive puncta throughout the entire brain. These cholesterol aggregates are evident 5 days after eclosion, but relatively scarce and mostly restricted to the cortical neuronal cell body layer at the surface of the brain. Forty-five days after eclosion, intense filipin puncta are abundant throughout the entire brain of Npc1a¹ mutant animals, including all central brain regions and the optic lobes.

At 5 days, Npc1a¹ mutants are indistinguishable from controls, displaying intact neuropil and cell bodies with no detectable evidence of vacuolization. however, retinal structures show clear signs of neurodegeneration, with the appearance of small vacuoles between clusters of undamaged ommatidia. In age-progressive studies at 5, 15, 30 and 45 days, neurodegeneration occurs first in the retina and spread progressively and more generally throughout the brain. Frontal sections of from day 15 Npc1a¹ animals maintain primarily intact neuropil and neuronal cell soma, with the occasional hole, whereas the retina exhibit clearly increased vacuolization. Day 30 Npc1a¹ mutants show a definite increase in central brain vacuolization with continuing deterioration of the retina. By day 45, frontal sections from Npc1a¹ mutants display extensive vacuolization and massive neural tissue loss in the central brain.

Npc1a¹ mutants exhibit loss of rhabdomere structure and membrane loss.

As early as day 1 after eclosion, before the onset of detectable movement defects or neurodegeneration in the central brain, neuron cell bodies of Npc1a¹ mutants already exhibit the formation of striking multi-lamellar bodies never observed in in the neuronal soma of age-matched controls. progressive accumulation of multi-lamellar bodies is observed in ever increasing numbers of neuronal cell bodies over time. By day 45, the cytoplasm of nearly every Npc1a¹ mutant neuron contains one or more clear multi-lamellar body or another extramembranous structure. Similar striking multi-lamellar bodies and multi-vesicular bodies are found in Npc1a¹ mutant retina, together with the specific loss of rhabdomere integrity. By day 45, Npc1a¹ mutants show extensive rhabdomere membrane intrusion in the IRS, as it deteriorates with age, with multi-lamellar bodies and multi-vesicular bodies commonly invading the IRS.

Neurodegeneration in Npc1a¹ mutants is not characterized by an increase in apoptosis.

Large mushroom body Npc1a¹ MARCM mutant clones (generated with control of Scer\GAL4^ey-OK107) show no consistent difference from wild-type clones in the maintenance of overall mushroom body morphology or the architecture of the extensive axonal projections. In age-progressive studies through day 50, the large mushroom body MARCM mutant clones show no detectable signs of apoptosis or cell loss that might indicate cell death or neurodegeneration.

Single cell Npc1a¹ mushroom body MARCM clones (under the control of Scer\GAL4^ey-OK107) show no age-dependent effects such as process loss or cell death.

Induced Npc1a¹ MARCM clones (generated with Scer\GAL4^elav-C155) randomly located through the central brain of day 30 animals clearly co-localize with accumulations of cholesterol aggregates and multi-lamellar bodies, whereas adjacent wild-type neurons maintain normal cytoarchitecture.

In young animals, both Npc1a¹ mutant and wild-type controls show rapid and sustained photoreceptor depolarisation during a light stimulus. Quantification of the sustained ERG amplitudes shows that Npc1a¹ mutants exhibit normal phototransduction early in life. Normal aging results in a slight reduction in mean ERG amplitude at day 40 compared with day 5. However, Npc1a¹ mutants exhibit a much greater age-dependent loss of phototransduction, with a 50% decrease in ERG amplitude compared with age-matched controls. Mutant animals exhibit a wide range of ERG amplitudes, with one response in the wild-type range and all others with little to no response to light stimulus.

Age causes a dramatic loss of synaptic on- and off-transients in Npc1a¹ mutant erg recordings compared with age-matched controls. Multi-lamellar bodies accumulate in intact cells and there is a high incidence of vacuoles in Npc1a¹ mutants. Npc1a¹ mutant photoreceptor terminals display very few capitate projection, consistent with a severe loss of synaptic transmission.

NPC1¹ mutants remain as first-instar larvae for a prolonged period before dying 90-192 hours AEL. The NPC1¹ first instar larvae show aberrant subcellular sterol accumulation. The first instar arrest can be partially rescued by feeding the larvae with 20E; when 20E is fed at 26 hours AEL, only 25% die during the first instar, 29% die during the transition to the second instar, 45% die in the second instar and 2% die in the third instar. When 20E is fed at 40 hours AEL, there is a weaker rescue. The first instar arrest can be significantly rescued by increasing the amount of cholesterol (which allows some survival to the pupal stage) or 7-dehydrocholesterol (which allows some survival to the adult stage) in the larval food. However, increasing the level of ergosterol, feeding larvae with desmosterol, or feeding with progesterone, does not rescue the larval lethality. Brains from 96 hour AEL NPC1¹ first-instar mutants show normal morphology with no evidence of neurodegenerative vacuoles.