Faculty Bibliography

Macroautophagy, a lysosomal pathway responsible for the turnover of organelles and long-lived proteins, has been regarded mainly as an inducible process in neurons, which is mobilized in states of stress and injury. New studies show, however, that macroautophagy is also constitutively active in healthy neurons and is vital to cell survival. Neurons in the brain, unlike cells in the periphery, are protected from large-scale autophagy induction because they can use several different energy sources optimally, receive additional nutrients and neurotrophin support from glial cells, and benefit from hypothalamic regulation of peripheral nutrient supplies. Due to its exceptional efficiency, constitutive autophagy in healthy neurons proceeds in the absence of easily detectable autophagic vacuole intermediates. These intermediates can accumulate rapidly, however, when late steps in the autophagic process are blocked. Autophagic vacuoles also accumulate abnormally in affected neurons of several major neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease, where they have been linked to various aspects of disease pathogenesis including neuronal cell death. The build-up of autophagic vacuoles in these neurological disorders and others may reflect either heightened autophagy induction, impairment in later digestive steps in the autophagy pathway, or both. Determining the basis for AV accumulation is critical for understanding the pathogenic significance of autophagy in a given pathologic state and for designing possible therapies based on modulating autophagy. In this review, we discuss the special features of autophagy regulation in the brain, its suspected roles in neurodevelopment and plasticity, and recent progress toward understanding how dysfunctional autophagy contributes to neurodegenerative disease.

Alpha-internexin, a neuronal intermediate filament protein implicated in neurodegenerative disease, coexists with the neurofilament (NF) triplet proteins (NF-L, NF-M, and NF-H) but has an unknown function. The earlier peak expression of alpha-internexin than the triplet during brain development and its ability to form homopolymers, unlike the triplet, which are obligate heteropolymers, have supported a widely held view that alpha-internexin and neurofilament triplet form separate filament systems. Here, we demonstrate, however, that despite a postnatal decline in expression, alpha-internexin is as abundant as the triplet in the adult CNS and exists in a relatively fixed stoichiometry with these subunits. Alpha-internexin exhibits transport and turnover rates identical to those of triplet proteins in optic axons and colocalizes with NF-M on single neurofilaments by immunogold electron microscopy. Alpha-internexin also coassembles with all three neurofilament proteins into a single network of filaments in quadruple-transfected SW13vim(-) cells. Genetically deleting NF-M alone or together with NF-H in mice dramatically reduces alpha-internexin transport and content in axons throughout the CNS. Moreover, deleting alpha-internexin potentiates the effects of NF-M deletion on NF-H and NF-L transport. Finally, overexpressing a NF-H-LacZ fusion protein in mice induces alpha-internexin and neurofilament triplet to aggregate in neuronal perikarya and greatly reduces their transport and content selectively in axons. Our data show that alpha-internexin and the neurofilament proteins are functionally interdependent. The results strongly support the view that alpha-internexin is a fourth subunit of neurofilaments in the adult CNS, providing a basis for its close relationship with neurofilaments in CNS diseases associated with neurofilament accumulation

Autophagy, a lysosomal pathway for degrading organelles and long-lived proteins, is becoming recognized as a key adaptive response that can preclude death in stressed or diseased cells. However, during development strong induction of autophagy in specific cell populations mediates a type of programmed cell death that has distinctive 'autophagic' morphology and a requirement for autophagy activity. The recent identification of autophagosomes in neurons in a growing number of neurodegenerative disorders has, therefore, sparked controversy about whether these structures are contributing to neuronal cell death or protecting against it. Emerging evidence supports the view that induction of autophagy is a neuroprotective response and that inadequate or defective autophagy, rather than excessive autophagy, promotes neuronal cell death in most of these disorders. In this review, we consider possible mechanisms underlying autophagy-associated cell death and their relationship to pathways mediating apoptosis and necrosis.

Degeneration of basal forebrain cholinergic neurons (BFCNs) contributes to cognitive dysfunction in Alzheimer's disease (AD) and Down's syndrome (DS). We used Ts65Dn and Ts1Cje mouse models of DS to show that the increased dose of the amyloid precursor protein gene, App, acts to markedly decrease NGF retrograde transport and cause degeneration of BFCNs. NGF transport was also decreased in mice expressing wild-type human APP or a familial AD-linked mutant APP; while significant, the decreases were less marked and there was no evident degeneration of BFCNs. Because of evidence suggesting that the NGF transport defect was intra-axonal, we explored within cholinergic axons the status of early endosomes (EEs). NGF-containing EEs were enlarged in Ts65Dn mice and their App content was increased. Our study thus provides evidence for a pathogenic mechanism for DS in which increased expression of App, in the context of trisomy, causes abnormal transport of NGF and cholinergic neurodegeneration.

Intracellular protein degradation decreases with age, altering the important balance between protein synthesis and breakdown. Slowly, protein accumulation events increase causing axonopathy, synaptic deterioration, and subsequent cell death. As toxic species accumulate, autophagy-lysosomal protein degradation pathways are activated. Responses include autophagic vacuoles that degrade damaged cellular components and long-lived proteins, as well as enhanced levels of lysosomal hydrolases. Although such changes correlate with neuronal atrophy in age-related neurodegenerative disorders and in related models of protein accumulation, the autophagic/lysosomal responses appear to be compensatory reactions. Recent studies indicate that protein oligomerization/ aggregation induces autophagy and activates lysosomal protein degradation in an attempt to clear toxic accumulations. Such compensatory responses may delay cell death and account for the gradual nature of protein deposition pathology that can extend over months/years in model systems and years/decades in the human diseases. Correspondingly, enhancement of compensatory pathways shifts the balance from pathogenesis to protection. Positive modulation of protein degradation processes represents a strategy to promote clearance of toxic accumulations and to slow the synaptopathogenesis and associated cognitive decline in aging-related dementias.

Phosphorylation of the carboxyl tail domains of the neurofilament heavy (NF-H) and middle molecular weight (NF-M) subunits has been proposed to regulate the axonal transport of neurofilaments. To test this hypothesis, we recently constructed mice lacking the extensively phosphorylated NF-H tail domain (NF-HtailDelta) and showed that the transport rate of neurofilaments in optic axons is unaltered in the absence of this domain [M.V. Rao, M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.A. Calcutt, Y. Uchiyama, R.A. Nixon, D.W. Cleveland, Gene replacement in mice reveals that the heavily phosphorylated tail of neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport, J. Cell Biol. 158 (2002) 681-693]. However, Shea et al. proposed that deletion of NF-H carboxyl-terminal region accelerates the transport of a subpopulation of neurofilaments based on minor differences between tail-deleted and control mice in our axonal transport analysis. Here, we present additional evidence that neurofilament transport rate is unchanged after deleting the phosphorylated NF-H tail domain, establishing unequivocally that the NF-H tail domain alone does not regulate the rate of neurofilament transport in optic axons in vivo. Possible roles for tail domains as cross-bridges between a neurofilament and its neighbors or other cytoskeletal elements is discussed

The identification of cathepsins in amyloid-beta plaques revealed broad dysfunction of the lysosomal system in Alzheimer's disease (AD). Coinciding with the discovery that proteolysis is required to generate the Abeta-peptide, these findings heralded an era of intense investigation on proteases in neurodegeneration. This review traces lysosomal system pathology from its early characterization to its origins within two pathways leading to the lysosome, the endocytic and autophagic pathways. An understanding has grown about how these two pathways are adversely influenced by normal brain aging and by genetic and environmental risk factors for AD, resulting in increased susceptibility of neurons to injury, amyloidogenesis, and neurodegeneration