The findings presented here reveal that enhancing endogenous protein homeostatic mechanisms within nerve tissue with a five month IF regimen significantly attenuates neuropathic behavioral and morphological phenotypes in a spontaneous mouse model of CMT1A. This dietary modulation is well-tolerated by TrJ mice and is associated with improved locomotor performance on the rotarod and increased grip strength. The benefits to the behavioral phenotype are paralleled by pronounced positive morphological changes within peripheral nerves, including increases in nerve fiber density, myelin thickness and axonal diameter. The dietary regimen partially alleviated the demyelinating phenotype of the neuropathy and supported the expression of myelin proteins. We observed a decrease in aberrant Schwann cell proliferation and in macrophage infiltration, both of which are characteristic features of CMT neuropathies (Misko et al., 2002
; Wang Ip et al., 2006
). Our studies indicate that the IF regimen enhanced multiple endogenous protective mechanisms within peripheral nerves, including the chaperone and the autophagy-lysosomal pathways, and prevented the inflammatory aspects and degenerative changes associated with the neuropathy.
While there have been major advances in understanding the genetics of hereditary neuropathies, treatment options for affected individuals are limited. The available transgenic and spontaneous animal models therefore serve as invaluable tools for studies aimed at understanding the cellular pathogenesis of the disease and to identify potential therapeutic targets. In a previous study, therapeutic administration of a progesterone antagonist between ages P5 and P49 in male PMP22 overexpressor transgenic rats reduced steady-state levels of PMP22 mRNA by 15%, which was sufficient to improve the disease phenotype (Sereda et al., 2003
). In PMP22 overexpressor C22 mice (Huxley et al., 1996
), ascorbic acid treatment between 2-5 months of age partially corrected the locomotor pathology by promoting the remyelination of axon fibers (Passage et al., 2004
). The precise molecular mechanism by which this anti-oxidant improved the neuropathic phenotype is unknown but likely involved decreasing the expression level of PMP22 (Kaya et al., 2007
). Ascorbic acid is now in clinical trials for CMT neuropathies (Pareyson et al., 2006
). Finally, a ninety day treatment with curcumin improved the motor performance and the histopathology of TrJ mice, when initiated in newborns (Khajavi et al., 2007
). Similar to our findings, these reports show that peripheral nerves of young mice respond to dietary supplements and modulations, although it is yet to be determined if these results will be reproducible in an older cohort of rodents. While the described regimen alleviates the neuropathy in affected mice, further studies will be required to determine how the human nervous system, particularly in patients with neurological diseases, may respond to long-term intermittent fasting.
We chose dietary restriction to alleviate the neuropathic phenotype of TrJ mice based on the ability of this approach to induce both chaperones and autophagy (Mizushima et al., 2004
; Steinkraus et al., 2008
), and knowledge concerning the subcellular trafficking of PMP22. The Wt PMP22, a substrate for proteasomal degradation, is an aggregation-prone hydrophobic protein with a low folding efficiency (Pareek et al., 1997
; Notterpek et al., 1999
; Sanders et al., 2001
). When one copy of the gene is mutated, the pool of PMP22 destined for the proteasome is increased, which leads to protein aggregate formation (Fortun et al., 2003
; Fortun et al., 2005
; Fortun et al., 2006
). The accumulation of misfolded PMP22 within Schwann cells interferes with the activity of the ubiquitin-proteasome system (Bence et al., 2001
; Fortun et al., 2005
), and serves as a nucleation site for the aggregation of protein chaperones, such as HSP70 and αB-crystallin (Ryan et al., 2002
; Fortun et al., 2005
; Fortun et al., 2007
). The entrapment of chaperones and proteasomal constituents within protein aggregates alters protein metabolic networks that function to ensure proper folding of newly-synthesized proteins, as well as the rapid degradation of misfolded proteins and short-lived regulatory molecules. Studies from various organisms indicate that such gradual decrease in chaperones by their recruitment to protein aggregates does not induce an increase in chaperone synthesis (Soti and Csermely, 2003
). Since myelinating Schwann cells are highly metabolically active, they are likely sensitive to such alterations which will impact their ability to maintain myelin homeostasis. Therefore, exogenous stimulation of chaperones to promote the folding and trafficking of PMP22 to the plasma membrane, and/or its redirection for proteasomal degradation may be a promising therapeutic avenue. Global induction of molecular chaperones can be achieved by brief exposure to stressful conditions, such as caloric restriction, or modulation of heat shock factor 1 (Morimoto et al., 1997
). We recently reported that pharmacologic induction of the heat shock response improves myelin synthesis and the processing of PMP22 in organotypic explant cultures from neuropathic mice (Rangaraju et al., 2008
). Similarly, here we show that increasing the endogenous levels of HSP70 within peripheral nerves is beneficial for myelination.
Autophagic protein degradation is an additional pathway that impacts the processing of PMP22 (Fortun et al., 2003
; Fortun et al., 2007
) and responds to dietary restriction (Mizushima et al., 2004
). Our results show that the IF regimen is effective in stimulating the autophagy-lysosomal pathway in peripheral nerves, which is reflected by reduction in poly-ubiquitinated substrates and induction autophagic markers (). Compounds that stimulate autophagic protein degradation are under investigation and may provide benefits in a variety of protein misfolding disorders (Rubinsztein, 2006
; Klionsky et al., 2008
). Rapamycin, a specific inhibitor of mTOR kinase, has been shown to regulate autophagy in cells from yeast to human (Inoki et al., 2005
) and can reduce the accumulation of poly-Q aggregates by routing the aggregates for lysosomal degradation via autophagy (Sarkar et al., 2008
). Nonetheless, as combined stimulation of HSPs and autophagy appears to be more beneficial for the processing of PMP22 than chaperone or autophagic stimulation alone (Fortun et al., 2007
), we chose the dietary approach which has the potential to activate both pathways. As our data reveals, this regimen has a pronounced influence on peripheral nerves and supported the maintenance of the differentiated Schwann cell phenotype (). We propose, that this benefit was a consequence of improved myelin stability and better axo-glial contacts, which is observed both biochemically and morphologically in TrJ mice on the regimen. Using the same study protocol, we detected similar morphological improvements in peripheral nerves of PMP22 overexpressor C22 mice (Huxley et al., 1996
) on the IF regimen, however the behavioral assessments are less consistent (data not shown). Because of the greater variability in the behavioral phenotypes in C22 mice, future studies will require a larger cohort of mice and/or more sensitive measures of motor performance.
Dietary restrictions are effective in increasing longevity and preventing degenerative changes associated with disease (Colotti et al., 2005
; Martin et al., 2006
; Sharma and Kaur, 2007
). For example, caloric restriction attenuated Aβ-deposition and decreased astrocytic activation in a transgenic rodent model of Alzheimer’s disease (Patel et al., 2005
). The mechanism underlying these beneficial effects are unclear, but likely involved multiple molecular pathways of neuroprotection, including the stress response, increased production of neurotrophic factors, ketone bodies and changes in glucose/insulin signaling pathways (Martin et al., 2006
). In comparison to the CNS, the influence of this intervention, particularly of the IF regimen, on peripheral nerve health and function has not been studied in detail. In a functional study, increased physical performance of mice on restricted diet was noted and attributed to behavior to avert risks to life (Ishihara et al., 2005
). As in our Wt mice, the regimen did not induce significant changes in motor behavior (), the improvement of neuropathic mice are rather due to benefits to neural function. Since early transplantation studies with tissue from TrJ mice unequivocally established that the neuropathy is due to a primary disorder of Schwann cells (Aguayo et al., 1977
; Perkins et al., 1981
), we focused our study on peripheral nerve proteins. Besides the myelin, stress and immunologic mechanisms, we investigated possible changes in the expression of sirtuins and neurotrophins within the nerve tissue, both of which have been linked to caloric restriction (Cohen et al., 2004
; Martin et al., 2006
). Additional pathways that could have been influenced by the IF regimen include ER-associated protein quality control mechanisms, changes in protein translation and proteasomal protein degradation (CITE??). With the available samples however our results are inconclusive so far. Finally, besides the direct influence of IF on the nerves, skeletal muscle-derived factors, such as neurotrophins may also have contributed to the observed improvement. In future studies, we plan on exploring this possibility.
Our results clearly show biochemical, morphological and behavioral improvements in TrJ neuropathic mice in response to a five month long dietary restriction. The benefits of this regimen are mediated in part by increased expression heat shock and autophagy-lysosomal proteins within peripheral nerves. These results support further work in developing and identifying compounds that stimulate protein homeostatic mechanisms, particularly the chaperone and autophagy-lysosomal pathways for treatment of CMT1A and other protein aggregate-associated diseases.