Neuronal pigmented autophagic vacuoles: lipofuscin, neuromelanin, and ceroid as macroautophagic responses during aging and disease

Abstract

The most striking morphologic change in neurons during normal aging is the accumulation of autophagic vacuoles filled with lipofuscin or neuromelanin pigments. These organelles are similar to those containing the ceroid pigments associated with neurologic disorders, particularly in diseases caused by lysosomal dysfunction. The pigments arise from incompletely degraded proteins and lipids principally derived from the breakdown of mitochondria or products of oxidized catecholamines. Pigmented autophagic vacuoles may eventually occupy a major portion of the neuronal cell body volume because of resistance of the pigments to lysosomal degradation and/or inadequate fusion of the vacuoles with lysosomes. Although the formation of autophagic vacuoles via macroautophagy protects the neuron from cellular stress, accumulation of pigmented autophagic vacuoles may eventually interfere with normal degradative pathways and endocytic/secretory tasks such as appropriate response to growth factors.

Fig. 1

Electron micrographs of human brain neuromelanin (NM) and lipofuscin (LF). a) NM autophagic vacuoles (AVs) displaying NM (electron dense matrix) and lipid droplets (*) in a substantia nigra dopamine neuron from a 78-year-old subject. Scale bar = 1 μm. The inset shows a 2.4-fold increased magnification of a single NM AV to clearly display a double membrane that delimits this organelle (arrow heads). b) AVs containing NM and lipid droplets (*) in a norepinephrine neuron of the locus coeruleus from a 81-year-old subject. The arrow indicates an expanse where double membrane can be observed indicating that the organelle is an AV. Scale bar = 1 μm. c) LF bodies in a norepinephrine neuron of locus coeruleus of the same subject. Scale bar = 500 nm.

Fig. 2

(a) Scanning electron microscopy image of dried neuromelanin (NM) pigment isolated from human substantia nigra (Bush et al. 2006) that appears to be composed of smaller spherical particles. Scale bar = 500 nm. (b and c) Atomic force microscopy confirms that NM aggregates are composed of approximately 30 nm diameter spheres, both after isolation (b) and within aggregated structures (c). Scale bar = 150 nm.

Fig. 3

Increased levels of neuromelanin (ng/mg wet tissue) in substantia nigra of human normal subjects during aging (Zecca et al. 2002). The values are expressed as mean ± SEM of 3–5 measurements.

Fig. 4

Induction of autophagic vacuoles (AVs) by l-DOPA. a–c) GFP-LC3 indicates l-DOPA induction of AVs. Ventral midbrain neuronal cultures derived from green fluorescent protein–LC3 (GFP–LC3) transgenic mice were treated with vehicle (cnt) or 100 μM l-DOPA for 48 h. Note that the control exhibits diffuse cytosolic fluorescence, whereas puncta (arrows) are visible in the l-DOPA-treated neurons. d–f) Monodansylcadaverine label is consistent with l-DOPA induction of AVs. Wild-type ventral midbrain neuron cultures were treated with 100 μM l-DOPA (e: 24 h, f: 1 week) or vehicle labeled with 50 μM monodansylcadaverine for 1 h. AVs appear to be absent in controls and are markedly enhanced by l-DOPA. Scale bars = 10 μm.