Sporadic short-term fasting, driven by religious and spiritual beliefs, is common to many cultures and has been practiced for millennia, but scientific analyses of the consequences of caloric restriction are more recent. Published studies indicate that the brain is spared many of the effects of short-term food restriction, perhaps because it is a metabolically privileged site that, relative to other organs, is protected from the acute effects of nutrient deprivation, including autophagy.17
We show here that this is not the case: short-term food restriction induces a dramatic upregulation of autophagy in cortical and Purkinje neurons. To our knowledge, this is the first demonstration that food restriction leads to in vivo neuronal autophagy. Our data extend recent reports in tissue culture systems,18,20
and have implications for individuals who, by choice or necessity, have limited food intake. Our findings also may have therapeutic implications, as outlined below. Autophagy is sometimes referred to as cellular “cleansing”, and our observations provide an attractive neuronal parallel to the organismal benefits that, historically, are perceived to derive from fasting.
In this study we describe a novel technique to identify and characterize autophagosomes in the tissues of GFP-LC3 mice, using vibratome sections and confocal microscopy. The changes that we report using this method are confirmed using the standard technique of TEM; this both validates the new approach, and confirms the conclusions that we have drawn therefrom. Herein, we make at least three novel observations. First, we identify and localize autophagosomes in cortical and Purkinje neurons of normal-fed mammals. Second, we show that, in these CNS neurons, short-term food restriction leads to a marked increase in the number of autophagosomes, together with changes in their physical characteristics. In principle, the observed changes could result either from increased production of autophagosomes, or from reduced fusion of autophagosomes with lysosomes. We believe that the former explanation is the more likely, because the activity of mTOR, a key protein that restrains autophagy, is reduced in the Purkinje cells of food-restricted mice (). Thus, our data favor the interpretation that the increased abundance of autophagosomes results from upregulation of the autophagy pathway in neurons, rather than from the blockade of autophagosome maturation, although some contribution from the latter cannot be excluded.
, we demonstrate that the changes that are induced by food restriction differ somewhat depending on the neuronal cell type. This is consistent with the recent proposal that autophagy pathways may differ among cell types, as a result of evolutionary adaptation to the specific physiological needs of the cell.14
Published studies in which autophagy was inhibited have shown that the outcome varies with cell or tissue type. For example, interruption of neuronal autophagy can lead to degenerative disease,1,2
but an equivalent block in pancreatic acinar cells causes no overt dysfunction.21
However, until now it has been thought that increased autophagy would result in phenotypically-similar changes in closely-related cell types. Here we show that this appears not to be the case in vivo. Following food restriction, we observed both similarities and differences in the size and distribution of autophagosomes in cortical neurons and Purkinje cells. The fact that the observed changes in autophagy differed between these two neuronal cell types suggests that a more extensive analysis is warranted, to determine the extent to which short-term food restriction induces autophagy—and, possibly, other changes—in the mammalian CNS.
Our observation that a brief period of food restriction can induce widespread upregulation of autophagy in CNS neurons may have clinical relevance. As noted above, disruption of autophagy can cause neurodegenerative disease, and the converse also may hold true: upregulation of autophagy may have a neuroprotective effect. For example, in vitro models have shown that starvation in neuronal cell lines can remove toxic molecules and damaged mitochondria from neurons.22–24
Other tissue culture studies, of mutant huntingtin and α-synuclein proteins (which are associated with Huntington disease and familial Parkinson disease respectively), have identified autophagy substrates that can be removed by drug-induced enhancement of autophagy. Most importantly, some neuroprotective effects of drug-enhanced autophagy also have been observed in vivo, in a D. melanogaster
model of Huntington disease.25
Finally, it has been suggested that intermittent fasting might improve neuronal function by means that are entirely independent of caloric intake, and may instead reflect an intrinsic neuronal response that is triggered by fasting;26,27
we speculate that the reported improvement of neuronal function may be related to the upregulation of autophagy that we show here. The above findings have encouraged the development of drugs that might enhance neuronal autophagy, thereby protecting against disease. Such drugs must: (i) be able to cross the intact blood-brain barrier; (ii) upregulate neuronal autophagy; and (iii) be harmless to the recipient. Food restriction is a simple, reliable, inexpensive and harmless alternative to drug ingestion and, therefore, we propose that short-term food restriction may represent an attractive alternative to the prophylaxis and treatment of diseases in which candidate drugs are currently being sought. However, caution is counseled, because studies in rat brain have suggested that chronic starvation might inhibit autophagy,28
an outcome that could damage, rather than protect, neurons.28,29