Our data demonstrate that mtDNA deletion mutations are higher in distal nerve segments in HIV-SN and are associated with impaired mitochondrial function and loss of mitochondrial respiratory chain complexes. Somatic mtDNA mutations, such as the common mtDNA4977
deletion, are considered markers of mtDNA damage in general that are known to accumulate in post-mitotic tissues during normal aging 11, 12, 14
. Increased levels of mtDNA mutations can also be found in some brain regions of patients with Parkinson’s 27
and Huntington’s disease 28
. These observations have led to the hypothesis that in neurodegenerative disorders, somatic mtDNA mutations accumulate with local variability that lead to a decrease in cellular energetic capacity and subsequent death of neurons in specific brain regions such as the substantia nigra. As an extension to this hypothesis, we studied spatial differences in mtDNA damage in proximal and distal parts of peripheral sensory nerves in HIV-SN and found that mtDNA deletion mutations are higher in distal nerve segments in patients affected in HIV-SN. Our analysis of the mitochondrial distribution showed that in cross-sections of human sural nerves around 84% of mitochondria are localized in axons rather than in Schwann cells, endothelial cells, fibroblasts or macrophages. Thus we conclude that increased levels of the common mtDNA4977
deletion are more likely to reflect changes in intra-axonal mitochondria rather than mitochondria in other cells (i.e. Schwann cells).
The human samples that we used for this study were from HAART-treated patients with end-stage HIV disease, thus HIV-SN in those cases is likely to be caused by both the use of antiretroviral agents and HIV infection. Examination of the specific role of antiretroviral toxicity versus HIV infection would be desirable but is impossible to achieve due to the fact that DRG and sural nerve samples obtained at post-mortem from untreated late stage HIV patients are not readily available in the era of HAART. Nucleoside reverse transcriptase inhibitors (NRTIs), the backbone of HAART, are well known to cause mitochondrial injury and subsequent neuropathy 1, 29
. Previous morphological studies on nerve biopsy specimens from patients treated with the dideoxycytidine (ddC) have noted ultrastructural abnormalities and loss of mitochondria in myelinated and unmyelinated axons of sural nerves 30
. NRTIs inhibit mitochondrial DNA polymerase-γ 31
, which is a key enzyme for replication and repair of the mitochondrial genome. Further, ddC, stavudine (d4T), and other NRTIs such as didanosine (ddI) are known to cause toxicity to primary neurons and neuronal cell lines in vitro by mitochondrial dysfunction 32, 33
. Recently, Zhu and colleagues demonstrated that in an animal model of HIV-1, the feline immunodeficeny virus (FIV) infection, ddI induced axonal injury and decreased mitochondrial COX-I expression in cultured feline DRG neurons and in DRG of FIV infected animals 34
. In accordance with these findings we observed loss of mitochondrial proteins and a decrease in expression of COX-I in sural nerves of patients affected with HIV-SN. Furthermore, we found that in macaques infected with SIV, mitochondrial-swelling assay was most abnormal in tenofovir-treated animals supporting the observation that NRTIs contribute to development of distal axonal degeneration by impairing mitochondrial function. Taken together, these data suggest that mitochondrial toxicity contributes to the pathogenesis of HIV-SN.
Clinical and pathological studies indicate that HIV-SN is a typical length-dependent “dying back” axonopathy, in which axonal degeneration starts in the distal end and continues in a centripetal manner. This pattern of involvement is not unique to HIV-SN and is common to most length-dependent peripheral neuropathies, including diabetic and most toxic neuropathies. This distal-to-proximal progression of axonal degeneration is responsible for what we clinically refer to as “stocking and glove” distribution of sensory and motor involvement. A long-standing hypothesis to explain length-dependency of most peripheral neuropathies has been the assumption that distal axons are akin to last field of an irrigation system getting the least “nutrients’ from the cell body 35
. What is unknown is the identity of “nutrients” from the cell body. Based on our observations we suggest that differences in health of mitochondria in proximal versus distal axons are responsible for the distal axonal degeneration seen in most peripheral neuropathies.
Can these observations also explain why most peripheral neuropathies, including HIV-SN, affect the unmyelinated small sensory fibers first? Although we don’t have a direct proof, we can speculate that differences in mitochondrial numbers in relation to the energy needs of an axon in myelinated versus unmyelinated fibers may underlie this selective vulnerability. We hypothesized that unmyelinated axons have lower numbers of mitochondria per axonal volume and thus at a disadvantage to meet local energy demands compared to myelinated axons. To our surprise, when we calculated number of mitochondria per axonal volume in human and primate sural nerves, the unmyelinated axons had almost twice the number of mitochondria compared to myelinated axons. This relative high density of mitochondria in unmyelinated axons has been observed in the optic nerve between unmyelinated and myelinated segments of the retinal ganglion neuron axons 36
. A review of the literature, however, suggests that despite a higher density of mitochondria, unmyelinated axons are at an “energy” disadvantage due to huge inefficiencies in the way they conduct action potentials 37–39
. When there is a local energy deficit (i.e. ATP depletion), Na+
ATPase failure can lead to reverse flow of calcium through the Na+
exchanger and result in axonal degeneration 40
. This mechanism has been proposed to underlie axonal degeneration seen in demyelinating lesions in multiple sclerosis (reviewed by Waxman 41
Compared to other cell types in the body, long peripheral nerves in humans provide a unique challenge to the biogenesis and maintenance of mitochondria. Although mitochondria are transported via fast axonal transport, movement of mitochondria is often bi-directional and the majority of mitochondria are stationary, often at nodes of Ranvier 42–45
. This results in an “effective” rate of axonal transport of mitochondria closer to the rate of slow axonal transport. If we assume that mitochondrial biogenesis occurs only in the DRG neuronal body, then intra-axonal mitochondria in nerves in human feet are 2–3 years “older” than the mitochondria in proximal segments of the same axon. The evidence for this hypothesis is indirect at this point. Our data demonstrate higher levels of mtDNA mutations in distal axons but does not provide direct data that these are in fact “older” mitochondria compared to their counterparts in proximal axons. Furthermore, future studies may show that mitochondrial biogenesis may occur within the axon during transport of mitochondria. One hypothesis to explain our data in view of such findings may be that mtDNA replication and mitochondrial biogenesis maybe inefficient and prone to mistakes and thus result in higher levels of mtDNA mutations in distal axons. Nevertheless, as aging is associated with mtDNA mutations, we can assume that distal axonal mitochondria are more likely to be dysfunctional with reduced capability to handle oxidative stress or meet the energy demands of the distal axonal terminal. In support of this hypothesis, we can look at the observation that axonal regeneration is less efficient in taller people. In a model of human nerve regeneration after capsaicin-induced denervation of the intraepidermal nerve fibers, height was an independent risk factor predicting the rate of reinnervation 46
. Of note, peripheral neuropathies, including HIV-SN, are more common among taller and older people 47–50
. This epidemiological observation supports the hypothesis that long axons are more vulnerable to degeneration from a variety of causes, likely because mitochondria in distal axons in older people have increased mtDNA modifications that hamper their ability to handle oxidative stress and local energy demands.
By examining only the common deletion mutation, we probably underestimated the total mtDNA damage seen in distal nerves. There are likely to be other mutations or modifications in mtDNA that are outside the common deletion region and still result in mitochondrial dysfunction. Future studies aimed at evaluating mtDNA damage and mitochondrial heteroplasmy with more sophisticated techniques can extend these observations. If our findings are confirmed in other peripheral neuropathies, it would help establish the central hypothesis that dysfunctional mitochondria in distal axons is a common mechanism to explain length-dependency of peripheral neuropathies. These findings will also provide a new direction for development of therapies aimed at maintaining mitochondrial health and function as a way to prevent and treat peripheral neuropathies and, perhaps, other neurodegenerative diseases.