Absence of the Cdk5 activator p35 causes adult-onset neurodegeneration in the central brain of Drosophila

Abstract

Altered function of Cdk5 kinase is associated with many forms of neurodegenerative disease in humans. We show here that inactivating the Drosophila Cdk5 ortholog, by mutation of its activating subunit, p35, causes adult-onset neurodegeneration in the fly. In the mutants, a vacuolar neuropathology is observed in a specific structure of the central brain, the 'mushroom body', which is the seat of olfactory learning and memory. Analysis of cellular phenotypes in the mutant brains reveals some phenotypes that resemble natural aging in control flies, including an increase in apoptotic and necrotic cell death, axonal fragmentation, and accumulation of autophagosomes packed with crystalline-like depositions. Other phenotypes are unique to the mutants, notably age-dependent swellings of the proximal axon of mushroom body neurons. Many of these phenotypes are also characteristic of mammalian neurodegenerative disease, suggesting a close relationship between the mechanisms of Cdk5-associated neurodegeneration in fly and human. Together, these results identify the cellular processes that are unleashed in the absence of Cdk5 to initiate the neurodegenerative program, and they provide a model that can be used to determine what part each process plays in the progression to ultimate degeneration.

Fig. 1.

Neurodegeneration in p35null central brain. (A) Absolute chronological and relative physiological aging process. To account for their shortened lifespan, p35 mutants (p35⁻; point 1) were compared with age-matched wild-type control w⁻ flies (point 2; black arrow for chronologically matched points) and also with control flies that had reached a similar position on their mortality curve (point 3; white arrow for physiologically matched points). (B,C) Young control and p35 mutant brains develop normally: C, cortex, Kenyon cells somatic region; ca, neuropil/calyx. (D,E) Vacuoles appeared randomly in old control brains but were accumulated in the MB area in old p35 mutants (red arrows). Vacuoles were present both in somatic and neuropil domains. Five brains for each genotype obtained in two independent experiments were sequentially sectioned. Thick (1 μm) plastic sections were stained with Toluidine Blue. Scale bar: 20 μm. (F–I) Actin cytoskeleton was deformed or deteriorated (red arrows) in old p35⁻ brains. Identical regions in the MB at the level covering Kenyon cell bodies, calyx (white dashed line), primary neurites and beginning of peduncle (blue dashed line) are shown in merged confocal images of whole-mount brain preparation. Optic slice: 1.6 μm. Scale bar: 20 μm.

Fig. 2.

Age-dependent progression of apoptotic cell death. Representative examples show TUNEL-positive nuclei in neurons (Elav positive; A–A″) and glia (Repo positive; B–B″) in p35 mutant brains. Note the weaker Elav signal in the apoptotic neuron in comparison with surrounding cells (white arrow in A–A″) and the weaker Repo signal in dying glial cell (white arrowhead in B–B″). Single confocal plan, optic slice: 1.6 μm. Scale bar: 10 μm. (C,D) The absolute number of TUNEL-positive cells per hemisphere is presented on the y axis (n≥6; mean ± s.e.m.). (C) Apoptosis in neurons. The pan-neuronal marker Elav was used to identify neurons (n≥6; mean ± s.e.m.) in three independent experiments. c, control. (D) Apoptosis in glia. The pan-glial marker Repo was used to identify glia (n≥6; mean ± s.e.m.). Time points under the chart show absolute age in days and refer to the position on the mortality curve; results are grouped according to the chronological points. For instance: 45-day-old p35 mutant flies reached 80% of mortality in population and were analyzed simultaneously with 45-day-old wild-type controls that reached only 20% in mortality.

Fig. 3.

Progressive increase in necrotic cell death. Necrotic cells were identified as propidium iodide (PI)- and Hoechst (HO)-positive nuclei. (A) Z-stack of confocal images through the MB area near the top of the brain (12 μm). Small trachea tubes surround the calyx (ca) of the MB and stain positive for PI (yellow arrow). Note the significant number of PI-positive cells due to physical damage: this portion of the brain was excluded from analysis. (B–B″) Example of a more-internal 5-μm-thick area included in analysis (outer white dotted line). PI-positive nuclei also exhibit bright HO signal and often have a larger diameter (hollow white arrow). Many cells have very low HO signal (white arrowheads). Necrotic cells were often clustered in both controls and mutants (dashed rectangles). Abundance and position of PI-positive cells implied that the majority of necrotic cells were neurons. Scale bar: 20 μm. (C) Increase in the percentage of necrotic cells with age; comparison of PI- and HO-positive versus HO-positive cells (n≥6; mean ± s.e.m.). Age-dependent time points are described in the legend to Fig. 1A and Fig. 2C,D. c, control.

Fig. 4.

Accumulation of autophagosomal organelles in old p35 mutant brains. Electron micrographs of MB neurons from control wild-type and p35 mutant brains at different time points. (A,B) Representative images of a middle-aged wild-type neuron (A) and young p35 mutant neuron (B). Boundaries of individual cells are highlighted by colored dotted lines. N, nucleus. (C,D) Age-dependent accumulation of autophagosomes (arrow) in both controls (C) and p35 mutants (D). Note the clusters of adjacent cells containing multiple ATG organelles. C′–F′ are high-magnification images of organelles highlighted in C–F, respectively. (E,F) Characteristic appearance of age-dependent multi-molecular depositions (arrowhead) in controls (E) and p35 mutants (F). Depositions with hexagonal symmetry were often located in close proximity to multilamellar structures. Scale bar: 500 nm. (G) Increase in the average number of autophagic organelles (AO) per neuron in old p35 mutants. Because APGs containing multilamellar structures represent the vast majority of all autophagic organelles that we observed in this project, we combined data for APGs, MLBs and APGLs. A minimum of 20 neurons per brain were analyzed. Values are mean ± s.e.m. (n≥3 brains). c, control.

Fig. 5.

Age-related neuronal degeneration. MARCM clones were analyzed in old flies above 80% of mortality of population. Images are Z-stack projections of sequential confocal slices covering the whole MB area. Brain midline is on the left, dorsal side is up. (A,A′) The first sign of degeneration in controls is when the axonal medial (m) lobe of γ-neuron appears thin and partially fragmented (arrowhead). (B,B′) Control γ-neuron with breaks in the proximal neurite (white dashed arrow) and completely missing axonal lobes (white dotted line). Note the end of the existing axon correlated with distal peduncle border (white hollow arrow). (C,C′) p35 mutant MB neuron has multiple breaks in the proximal neurite (dashed arrow), and the dorsal and medial axonal lobes (arrows). Note that the peduncle part of the proximal axon exhibits a reduced amount of fragmentation. Present parts of the proximal neurite (pn), peduncle (p), dorsal (d) and medial (m) axonal lobes are highlighted with a single dashed line. Missing links of neurites are highlighted with fine white dotted line. Other non-MB neuronal types labeled with GFP are marked with a star. Scale bar: 30 μm.

Fig. 6.

Age-dependent changes in morphology of p35 mutant neurons. MARCM clones were analyzed in young (5-day old) and old flies above 80% of mortality in population. Images are Z-stack projections of the whole MB area. For orientation and labeling details, see legend to Fig. 5. Young (A) and old (B) controls and young p35 mutants (C) have normal morphology. Old p35 mutants (D) develop swelling (white arrow and bracket) in the area of AIS compartment (white rectangle). Scale bar: 30 μm. (D′,E) Higher magnification showing details of swellings. (D′) Boxed area in D; note bright GFP clog. (E) Example of axonal swelling from another brain. Position of the axon is highlighted by a white dotted line; dendrites are marked as ‘de’. Scale bar: 10 μm.