22 April 2006. When it comes to cleaning house, neurons are no slouches. Considering the number of neurodegenerative diseases that are triggered by the accumulation of protein garbage, it is clear that these cells cannot tolerate messes. For quick pickups, the ubiquitin-proteasome system tags and destroys some proteins. But, as two papers in this week’s Nature online demonstrate, keeping neurons spic, span, and healthy, requires sweeping with a broader broom.
That broom is autophagy, the process of bulk protein degradation, where bits of cytosol, including some organelles, are incorporated into lysosomes. Work from two independent Japanese groups shows that disrupting autophagy, by knocking out either of two genes critical to the process, causes widespread neurodegeneration in mice. In neurons, loss of this constitutive clearance system leads to an age-dependent accumulation of ubiquitinated proteins in the cytosol and the formation of inclusion bodies. Together, the two papers, one from the lab of Noboru Mizushima and the other from Keiji Tanaka, both at the Tokyo Metropolitan Institute of Medical Science, reveal that autophagy is essential to the normal function and health of neurons. Their work highlights the role of this housekeeping pathway in neurodegenerative disease, where it offers a potent counterforce to the accumulation of toxic proteins and formation of inclusion bodies (see ARF related news story).
Cells use two major protein degradation systems for quality control of new protein synthesis and disposal of old or damaged proteins. The ubiquitin-proteasome system removes individual proteins that have been marked for degradation. The other system, autophagy, relies on vesicles to engulf portions of cytoplasm and deliver them to lysosomes. Macroautophagy, one type of “self-eating,” is the main mechanism cells have for turning over long-lived proteins and organelles. Autophagy is activated in Huntington, Parkinson, and Alzheimer diseases, presumably as a protective response to the accumulation of toxic proteins or aggregates (see ARF related news story and Yu et al., 2005).
Autophagy is an important cell homeostatic process, and knockout of either of two key genes in the autophagy pathway, Atg5 or Atg7, causes perinatal death in mice. To look at the function of autophagy specifically in neurons, the researchers, working with collaborators across Japan, used the nestin promoter and a Cre/lox system to produce live mice with either Atg5 (in the case of the Mizushima group) or Atg7 (by Tanaka and colleagues) deleted specifically in neurons. They showed that most neurons lacked Atg proteins and autophagic activity.
Both sets of mice revealed strikingly similar phenotypes of neurodegeneration and inclusion body formation starting early in life. By three weeks, the Atg5-lacking mice displayed problems with balance, coordination, and strength, including abnormal walking, limb clasping defects, and failure to balance on a rotating rod for even a short time. Atg7 knockouts had very similar progressive deficits, but were even sicker, showing lowered survival by 4 weeks of age, and most died by 28 weeks old.
For the Atg5 knockouts, the gross anatomy of their brain was normal, but on closer inspection the researchers, led by first author Taichi Hara, found evidence of neuron loss, most prominently in the cerebellar Purkinje cells, and apoptosis (in neighboring granular layer cells). Hippocampal pyramidal cells were also affected, and throughout the brain there was evidence of axonal swelling. In the case of the Atg7 knockout, joint first authors Masaaki Komatsu and Satoshi Waguri documented a more pronounced neuronal loss in both the cerebellum and cerebral cortex, and saw apoptosis of neurons in the cortex.
Staining of brain tissues with anti-ubiquitin antibodies revealed a dramatic phenotype of progressive accumulation of large, ubiquitin-positive inclusion bodies throughout the brain. The aggregates of misfolded proteins were seen in neurons but not glial cells, consistent with the conditional knockout of the two genes and loss of autophagy only in neurons. In the Atg5 knockout, inclusion formation was preceded by an increase of diffuse ubiquitin staining, starting in embryonic cells. From this data, Hara et al. conclude that the primary cellular phenotype of Atg5-deficient neurons is the accumulation of diffuse abnormal cytosolic proteins, with inclusion body formation occurring later. Their results suggest that the main function of autophagic vesicles is to take up and destroy diffuse proteins, not to gobble up inclusion bodies, as has been previously suggested. Formation of inclusion bodies follows on the inadequate clearance of the soluble proteins. Experiments in mice with an inducible liver-specific loss of Atg5 supported this idea, as those mice also demonstrated a rapid build-up of diffuse cytosolic ubiquitinated proteins, followed by formation of inclusion bodies after induction. Komatsu et al. showed that Atg7 deficiency did not affect proteasome function, so that the accumulation of ubiquitinated proteins was a direct result of ineffective autophagy.
If failure of autophagy leads to the accumulation of ubiquitinated proteins, the formation of inclusion bodies, and neurodegeneration in otherwise normal neurons, what about its role in human neurodegenerative disease? From a number of studies, it’s clear that this constitutive housecleaning pathway has an important role to play in conditions like Parkinson and Huntington diseases, which feature polyubiquitinated inclusions. Autophagy also contributes to the metabolism of amyloid-β peptides in Alzheimer disease. Attempts to boost autophagy using the drug rapamycin show some beneficial effects in enhancing clearance of toxic proteins in models of neurodegeneration (see ARF related news story and Berger et al., 2006). Going forward, it will be essential to sort out the contributions of autophagy versus proteasomal degradation pathways (see ARF related news story) in diseases where cells are fighting off the assault of mutated or misfolded proteins.—Pat McCaffrey.
Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R, Yokoyama M, Mishima K, Saito I, Okano H, Mizushima N. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature Advance Online Publication, April 20, 2006. Abstract
Komatsu M, Waguri S, Chiba T, Murata S,Iwata J, Tanida I, Ueno T, Koike M, Uchiyama Y, Kominami E, Tanaka K. Loss of autophagy in the central nervous system
causes neurodegeneration in mice. Nature Advance Online Publication, April 20, 2006. Abstract