Mitochondrial DNA deletions parallel age-linked decline in rat sensory nerve function

Mitochondrial DNA deletions parallel age-linked decline in rat sensory nerve function

Neurobiology of Aging 22 (2001) 635– 643 www.elsevier.com/locate/neuaging Mitochondrial DNA deletions parallel age-linked decline in rat sensory ner...

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Neurobiology of Aging 22 (2001) 635– 643

www.elsevier.com/locate/neuaging

Mitochondrial DNA deletions parallel age-linked decline in rat sensory nerve function Phillip Nagleya,*, Chunfang Zhanga, Maria L.R. Lima, Merhi Merhib, B. Elise Needhama, Zeinab Khalilb a

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia b National Ageing Research Institute, University of Melbourne, Parkville, Victoria 3052, Australia Received 20 November 2000; received in revised form 19 February 2001; accepted 23 February 2001

Abstract In rats, the function of sensory nerves in the hind limb declines significantly with age. Normally aging rats and rats treated neonatally with capsaicin were studied here. Quantification of vascular response and substance P in young (3 months) and old (24 months) rats showed additive effects of age and capsaicin treatment. The levels in dorsal root ganglion of a particular deletion in mitochondrial DNA (mtDNA4834) were about 300-fold higher in old compared to young rats. Capsaicin treatment had no significant effect on mtDNA4834 abundance. Dorsal root ganglia of old (but not young) rats were found to contain a spectrum of multiple deletions. The abundance of mtDNA4834 in dorsal root ganglia from individual rats correlated strongly with their decline in vascular function, even where vascular responses were systematically depressed due to prior capsaicin treatment. One possibility is that mitochondrial DNA mutations directly lead to functional decline at mitochondrial and tissue levels. Alternatively, loss of mitochondrial DNA integrity and physiological decline may be consequences of the same factor, such as oxidative stress. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Mitochondrial DNA; Multiple deletions; Dorsal root ganglia; Capsaicin; Animal model; Polymerase chain reaction; Vascular response; Substance P; Sciatic nerve; Rat

1. Introduction The ageing process is a multi-causal phenomenon. One concept under wide consideration attributes the decline of function in many tissues to respiratory deficiencies caused by progressive accumulation of mitochondrial DNA (mtDNA) mutations with age [26,35,38,45]. Thus, in humans the abundance of mutant mtDNA molecules (mtDNA4977) carrying the so-called “common” 4977 bp deletion has been shown to have a strong positive correlation with age in many tissues [reviewed in 27,36]. Moreover, the accumulation of multiple mtDNA deletions, representing a broad spectrum of independent mutagenic events [12], has also been shown to have a distinct ageassociated relationship in several human tissues [23,28,32]. Studies of overt mitochondrial diseases have revealed compelling links between the prevalence of mtDNA mutations * Corresponding author. Tel.: ⫹61-3-9905-3735; fax: ⫹61-3-99054699. E-mail address: [email protected] (P. Nagley).

and pathophysiological changes in tissue function [29,46]. However, there is a paucity of data concerning directly ascertained correlations between the abundance of mtDNA mutations and the decline in the physiological function of human tissues with normal aging. One experimental approach to investigate the applicability, if any, of such relationships in aging mammals is to use an animal model such as the laboratory rat. A “common” 4834-bp deletion in rat mtDNA, similar in size and location to those in human mtDNA4977, occurs between a pair of 16-bp direct repeats located in rat mtDNA at nucleotide positions (nt) 8103– 8118 and 12937–12952, respectively [9,27]. Rat mtDNA molecules carrying this deletion (mtDNA4834) have been shown to accumulate with age in a number of tissues [6,7,8,9,27,51]. The prevalence of mtDNA4834 is generally less than that of mtDNA4977 in corresponding human tissues, notably in skeletal and cardiac muscle [51]. Multiple deletions of mtDNA in rat tissues have not been widely encountered [51] with the notable exception of brain [44,49] and kidney [51]. The rat hind-limb system [21], including the primary

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afferents of the sciatic nerve and their cell bodies in the dorsal root ganglion (DRG), represents an attractive system for integrating analyses of mtDNA mutations with explicitly demonstrated age-associated physiological decline. A major part of the normal inflammatory vascular response in peripheral tissue is dependent upon intact unmyelinated primary afferent sensory innervation [14,15,22,30,40]. These nerves mediate early components of the neurogenic inflammatory response [17,18] and subsequently initiate the process of tissue repair [14,37]. These effects are mediated by neuropeptides like substance P (SP) that are synthesized in DRG cells, transported, and released from both central and peripheral terminals of these nerves. There is a decline in the function of the primary afferent unmyelinated sensory nerves in aged rats [21] with a consequential decrease in the capacity to maintain appropriate inflammatory vascular responses, which correlates with a decline in wound repair efficacy [1,19]. Significantly, these age-associated phenomena are manifested not only in rats [19,21] but also in humans [1,20]. Such age-dependent neuronal decline in old animals can be pharmacologically mimicked [21] in young rats treated neonatally [24] with the neurotoxin capsaicin, that selectively destroys the unmyelinated primary afferent sensory nerves [16]. Capsaicin-treated animals undergo permanent loss of the bulk of their unmyelinated primary afferent sensory nerves and depletion of their sensory neuropeptides. Consequentially, they show reduced inflammatory vascular responses, poor wound healing [19] and diminished skin integrity [15]. We investigated here the relationships between the agerelated physiological decline of neuronal tissues in the rat hind limb (assessed by vascular response to sensory nerve activation and SP content in the DRG) and the prevalence of mtDNA mutations in the DRG, comparing young and old rats. Using a capsaicin-treated cohort of rats as a reference, we were able to define an age-dependent accumulation of mtDNA4834 that correlated very strongly with loss of vascular response at the extremity of the hind limb.

2. Methods 2.1. Animals and tissues Outbred male Sprague-Dawley were used, with average weights of 250 –350 g for young rats (3 months), 600 –700 g for old rats (24 months). Anaesthesia was induced with sodium pentobarbitone (60 mg/kg, i.p.) and maintained by supplementary injections. During experimental procedures, body temperature was maintained at 37°C. Animals were sacrificed by barbiturate overdose at the completion of experiments. After dissection, DRG were weighed and then frozen in hexane/liquid nitrogen for storage at ⫺70°C until required. All experimental procedures on animals were approved by the Animal Ethics Committee of the Royal Melbourne Hospital.

2.2. Neonatal capsaicin treatment Neonatal rats were treated on the second day of life with a single subcutaneous injection of capsaicin (50 mg/kg) [19,21]. Subsequent experiments described below were performed at the age of three or 24 months. Efficacy of prior drug treatment was confirmed by applying a drop of capsaicin (0.1%) to the eye and recording the subsequent number of eyewipes. Rats were considered capsaicin-denervated if the number of eyewipes was less than 25% of controls. 2.3. Blister induction and antidromic stimulation of sensory nerves A blister was induced on the hindpaw of the anaesthetised rat using a vacuum pressure of 40 kPa [37]. After exposure of the sciatic nerve at the mid-thigh region, the nerve was cut and the distal portion placed over platinum electrodes and immersed in a warm oil pool in an enclosure formed from the skin flaps of the wound. The electrodes were fixed in such a position that electrical leakage to adjacent muscle and tissue structures was minimised. The surface epidermis of the blister was then removed and a perspex chamber (with inlet and outlet ports) secured over the blister base. Ringer’s solution was perfused over the blister surface continuously during subsequent procedures [21,33,41]. Activation of the sensory fibres was achieved with a Grass stimulator at 20 V, 15 Hz, 2 ms for a duration of 1 min. These parameters have been previously used to stimulate efferent C-fibre responses [24,33,34] evoking an immediate increase in local blood flow. The time elapsed from the start of the blister induction until sciatic nerve stimulation did not exceed 60 min. 2.4. Measurement of cutaneous blood flow A laser Doppler flowmeter probe was positioned vertically over the exposed blister in the hindpaw via the central port of the perspex chamber. The changes in relative blood flow (as determined by changes in red cell flux) following electrical stimulation of the sciatic nerve were continuously displayed on a chart recorder. Raw data were evaluated by computing the area under the stimulation-evoked response curve for a post-stimulation period of 20 min. All measurements were made relative to a stable baseline obtained prior to nerve stimulation. At the completion of the stimulation, sodium nitroprusside (100 ␮M) was perfused over the blister base, to control for overall smooth muscle reactivity within the groups. 2.5. Extraction and analysis of substance P DRG from individual rats were homogenized in acetic acid (2 M) using a Polytron homogenizer (Kinematica, Switzerland) and further extracted using the acetone/HCl method [4]. The technique used to quantify SP in tissue

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extracts is modified from that of ref. 48. The tracer used was iodinated [tyr8]-SP, prepared using the chloramine T method. Separation of the bound and free tracer was achieved by addition of a dextran-coated charcoal mixture. The RMSP-1 antiserum (Auspep, Australia) was used at a final dilution of 1:300,000. This antiserum has been previously shown to be highly specific for SP [2]. Absolute sensitivity of the assay was 3 pg/tube. The inter-assay variability coefficient was 5.5%. SP concentrations were calculated as molar amount of SP per wet tissue weight. 2.6. DNA isolation, polymerase chain reaction and cloning Total cellular DNA was extracted from rat DRG, polymerase chain reaction (PCR) carried out and products analyzed electrophoretically as described [50,51]. Synthesis and nomenclature of oligonucleotide primers were as described [51]. A 416-bp PCR product encompassing the breakpoint of mtDNA4834 was amplified using the primer pair rL7868[27] plus rH13117[19] and cloned at the SmaI site of pUC18 as described [50]. The recombinant plasmid was denoted pCZ56. 2.7. Quantification of mtDNA4834 The abundance of mtDNA4834 in rat DRG was quantified following the procedure previously used to quantify specific mtDNA deletions in human tissues [52]. This involved the use of a set of varied inputs of pCZ56 as external reference for PCR. A 235-bp region common to both non-mutant mtDNA and mtDNA4834 was amplified using primers rL7868[27] and rH8102[19] for 30 PCR cycles (inputs of pCZ56 DNA ranged from 0.001 to 2 ng), whereas a 416-bp region spanning the deletion breakpoint was amplified using the primer pair rL7868[27] plus rH13117[19] for 35 PCR cycles (inputs of pCZ56 DNA ranged from 5 ⫻ 10⫺7 to 1 ⫻ 10⫺4 ng). From comparisons of the yield of PCR products from these external standards with those derived from appropriate inputs of tissue-derived DNA (containing total mtDNA equivalent to 0.1 ng of pCZ56 DNA), the abundance of mtDNA4834 molecules was quantified in each tissue extract (expressed as a percentage of total mtDNA). Each such measurement was carried out at least twice, with identical results for each measurement. The sensitivity of detection was such that the lowest percentage of mtDNA4834 which could be detected was 0.00005% of total mtDNA. 2.8. Analysis of multiple deletions Two pairs of primers [51], each spanning a longer segment of mtDNA than the pair used to analyse mtDNA4834, were employed to detect multiple deletions in mtDNA of rat DRG under the Hot Start PCR conditions previously described [51]. The small expansion primer pair rL7868[27]

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plus rH13610[24] spans a 5743-bp region of the mtDNA genome, whereas the widely spaced primer pair rL7868[27] and rH16110[26] spans 8243 bp of mtDNA. 2.9. Statistical analysis Statistical significance was determined using the paired Student’s t test or linear regression analysis. Results are expressed as the mean ⫾ SEM.

3. Results 3.1. Effects of age and capsaicin treatment on sensory nerve responses Following electrical stimulation of the sciatic nerve in young untreated rats, a vasodilatation response was obtained that was maintained for about 10 –15 min then gradually declined. The mean area under the curve for young controls was 18.1 ⫾ 0.8 cm2. The vascular response in old rats was significantly reduced (10.1 ⫾ 0.8 cm2) ( p ⬍ 0.001) reaching only 56% of the response in young animals (Fig. 1A). The responses in both young and old capsaicintreated rats (7.9 ⫾ 0.5 cm2 and 4.9 ⫾ 0.5 cm2, respectively) were also significantly reduced ( p ⬍ 0.001) compared to their age-matched controls, as were those of the old capsaicin-treated rats compared to their young treated counterparts. This suggests that both age and capsaicin-treatment contribute additively to the impairment of vascular response. The vasodilator sodium nitroprusside was perfused over the blister base following the stimulation responses. The responses to sodium nitroprusside were not significantly different between young untreated (25.3 ⫾ 2.3 cm2), young capsaicin-treated (22.8 ⫾ 3.5 cm2), old untreated (28.5 ⫾ 2.3 cm2) and old capsaicin-treated (24.5 ⫾ 4.5 cm2) groups. This indicates that vascular smooth muscle function, as such, is not subject to age-related or capsaicin-induced debilitation. 3.2. Substance P content of dorsal root ganglia The level of SP in DRG was determined by quantifying SP-like immunoreactivity. Similarly to those of the vascular responses, the data (Fig. 1B) indicate the decline in SP content of DRG occurs cumulatively as a result of both aging and capsaicin treatment. In DRG of young untreated rats the SP content was 105.7 ⫾ 8.2 fmol/mg. There was a significant reduction in SP-like immunoreactivity in DRG of old untreated rats (55.3 ⫾ 4.9 fmol/mg) and young and old capsaicin-treated rats (31.3 ⫾ 4.3 and 17.9 ⫾ 2.4 fmol/ mg, respectively).

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Fig. 1. Sensory nerve response and sensory peptides in DRG as a function of age and capsaicin treatment. (A) Vascular response to antidromic electrical stimulation of the sciatic nerve was measured on the footpad using a laser Doppler flowmeter. Units represent the area under the stimulation evoked response curve (cm2). Groups of rats were as follows: young untreated (n ⫽ 11), old untreated (n ⫽ 11), young capsaicin-treated (n ⫽ 6), old capsaicin-treated (n ⫽ 6). (B) SP content was determined by quantifying SP-like immunoreactivity in DRG of individual rats. Groups of rats were as follows: young untreated (n ⫽ 6), old untreated (n ⫽ 6), young capsaicin-treated (n ⫽ 7), old capsaicin-treated (n ⫽ 7). Comparisons between groups showing highly significant differences ( p ⬍ 0.001) are as follows: *, old untreated and young untreated; **, young treated and young untreated; ***, old treated and old untreated. The symbol # indicates the comparison between old and young capsaicin-treated rats to be significant ( p ⫽ 0.0012 for vascular response; p ⫽ 0.011 for SP content).

3.3. Age-associated accumulation of mtDNA4834 in rat DRG The abundance of mtDNA4834 (expressed as percentage of total mtDNA) was measured in DRG of 41 rats. The mean values for each of the four groups analyzed (Fig. 2)

Fig. 2. Abundance of mtDNA4834 in DRG of rats. An external standard PCR method was used to quantify mtDNA4834 in DRG of rats in four groups: young untreated (n ⫽ 9), old untreated (n ⫽ 16), young capsaicintreated (n ⫽ 7) and old capsaicin-treated (n ⫽ 9). Levels of mtDNA4834 in 5 of the 9 young untreated rats were below the limit of detection (0.00005% total mtDNA). For the purposes of calculating a mean value these were assigned arbitrary values by random number generation [28,52]. Comparisons between groups showing significant differences are as follows: *, old untreated and young untreated ( p ⫽ 0.0026); **, old treated and young treated ( p ⫽ 0.016).

show a clear effect of age on the abundance of mtDNA4834, but the effect of capsaicin, as such, appears insignificant. The deletion was detected in four of the nine young untreated rats studied. The abundance of mtDNA4834 in the young rats ranged from below the limit of detection (0.00005% of total mtDNA) to measured values of 0.00011%. It was possible to calculate a mean value for this group (0.000061 ⫾ 0.000008% of total mtDNA), using randomly generated numbers [28,52] to arbitrarily assign values for those samples containing undetectable levels of mtDNA4834 (see legend to Fig. 2). In contrast, mtDNA4834 was detected and quantified in DRG of all 16 old untreated rats tested, the abundances ranging from 0.006% to 0.055% of total mtDNA with an average of 0.016 ⫾ 0.004%. The differences in mean abundance of mtDNA4834 between old and young rats is highly significant ( p ⬍ 0.005). In DRG of the seven young capsaicin-treated rats studied, the abundance of mtDNA4834 in six rats ranged from 0.000055% to 0.00015% of total mtDNA; mtDNA4834 in DRG of the remaining rat comprised 0.01% of total mtDNA. The mean value (incorporating the outlier) is 0.0015 ⫾ 0.0013% (Fig. 2), but discounting the outlier generates a calculated mean of 0.000084% (close to that of the young untreated rats). The nine old capsaicin-treated rats ranged from 0.006% to 0.077% in their DRG levels of mtDNA4834, with a mean abundance of 0.033 ⫾ 0.01% of total mtDNA. Comparing the old and young capsaicin-treated rats, the abundance of mtDNA4834 in old rats was found to be significantly higher than that in the young rats ( p ⬍ 0.02) even when the outlier was included in the analysis. However, there was no significant difference in mtDNA4834 levels between young untreated and capsaicin-treated rats, or between old untreated and capsaicin-treated rats.

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Fig. 3. Multiple mtDNA deletions in DRG of young and old untreated rats. (A) PCR products amplified with primer pair rL7868[27] plus rH13610[24] for 30 cycles. (B) PCR products amplified with primer pair rL7868[27] plus rH16110[26] for 35 cycles. In each panel, PCR products amplified from DRG of young (lanes 1–9) and old (lanes 10 –25) untreated rats are displayed. Lane M, ␭ DNA digested with EcoRI and HindIII, as size markers; the sizes of some bands are indicated at the left. In panel A, the positions of PCR products of full-length mtDNA (5743 bp) (F) and mtDNA4834 (909 bp) (D) are indicated.

3.4. Multiple mtDNA deletions in DRG of old rats In the analyses of mtDNA4834 described above, using the primer pair rL7868[27] and rH13117[19], the two major PCR products observed on the gels were the full-length product (5250 bp) of amplification from non-deleted mtDNA templates and the 416-bp product of amplification from mtDNA4834 templates (not shown). A small number of relatively weak additional bands was observed on these gels, some appearing in both young and old DRG samples, whilst others were only seen in old samples. The latter are suggestive of age-related multiple deletions. Two further primer pairs were used to examine the range and relative intensities of PCR products potentially representing multiple mtDNA deletions in rat DRG. First, the small expansion primer pair rL7868[27] and rH13610[24] was used, in which the downstream primer is shifted 493 bp

from the position of rH13117[19], allowing a slightly enlarged window of deletions to be detected. It can be seen from Fig. 3A that only the full-length 5743-bp product was amplified from tissues of the young untreated rats whereas numerous PCR products smaller in size than 5743 bp were amplified from those of the old rats. Each of the samples corresponding to the DRG of 16 old untreated rats (lanes 10 –25 of Fig. 3A) showed this full length PCR product, the 909-bp product of amplification of mtDNA4834 and a multiplicity of other bands ranging in size from about 0.2 kb to 3 kb. These PCR products are considered to represent deletions of 3.7 kb to 5.5 kb, a unique set of which are seen in each of the old DRG samples. Similar results were obtained with the capsaicin-treated young and old rats (data not shown). Second, when the widely spaced primer pair rL7868[27] plus rH16110[26] was used, PCR products ranging in size

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between 0.7 kb and 3 kb were amplified in samples of old rats, representing deletions from 5.2 to 7.5 kb in size (Fig. 3B). Note that the PCR product of mtDNA4834 (expected size about 3.4 kb) was not observed in Fig. 3B due to its relative inefficiency of amplification in competition with shorter amplification products of mtDNA templates carrying larger deletions [50]. As above, the set of multiple deletions in each old rat DRG differed from that of every other old rat, but no multiple deletions were detectable in DRG in young rats. The full-length product of 8243 bp was not appreciably amplified under the PCR conditions used here. These results demonstrate the age-associated accumulation of multiple deletions of mtDNA in rat DRG. However, it has not been possible to explicitly quantify the prevalence of multiple deletions observed in this manner. The assignment of these novel variable bands to represent a set of deletions (rather than artefacts of PCR) is based on extensive prior experience [36] of sequencing such bands recovered from gels for samples from both rats [44,51] and humans [50,51,53]. Hot Start PCR was explicitly used in tests of multiple deletions to avoid possible artefacts [51]. Finally, the bands appeared only in DRG extracts from old (but not young) rats. 3.5. Correlation between mtDNA4834 abundance and decrease in vascular response A critical test was to correlate the quantified abundance of mtDNA4834 in individual rats with the vascular response observed for that same rat. Vascular response data were available for 25 rats in which the abundance of mtDNA4834 in DRG was measured. The vascular activity for each rat was expressed as a percentage relative to the mean of that of the five young untreated rats in this set. Data are plotted against the natural logarithm of the abundance of mtDNA4834 for the three remaining groups (Fig. 4). There is a striking correlation between these two parameters for the old untreated rats ( p ⫽ 0.000005), suggestive of a strong relationship between accumulation of mtDNA deletions and loss of physiological function. The correlations for the two sets of capsaicin-treated rats are also highly significant (old, p ⬍ 0.005; young, p ⬍ 0.05). Indeed, the plots for these two groups suggest that they may be part of a continuous relationship across the two age groups of treated rats. The rightmost point for the young capsaicin-treated rats represents that of the outlier mentioned above in relation to Fig. 2, and it can be seen to fit neatly into the data set of the corresponding old rats. It was not useful to plot the results for the five individual young untreated rats because their measured vascular responses for all were close to the mean of 100%. Moreover, in one such rat the abundance of mtDNA4834 was below the limit of detection, obviating its graphical representation in a plot such as that in Fig. 4. The data in Fig. 4 show that the old capsaicin-treated rats had, on average, a greater decrease in vascular response

Fig. 4. Correlation between vascular response and abundance of mtDNA4834 in individual rats. Data are shown only where the same individual animals were available for both measurements and were obtained for old untreated (n ⫽ 9, r2 ⫽ 0.96, p ⫽ 0.000005), young capsaicintreated (n ⫽ 7, r2 ⫽ 0.63, p ⫽ 0.03) and old capsaicin-treated (n ⫽ 4, r2 ⫽ 0.99, p ⫽ 0.004) rats. The control for vascular response data is the mean value for a parallel group of five young untreated rats. The abundance of mtDNA4834 (% total mtDNA) is expressed as the natural logarithm.

compared to that of the old untreated group (means of 47% and 23%, respectively) (cf. Fig. 1A). Nevertheless, the abundances of mtDNA4834 in the old capsaicin-treated rats were in the same range as that observed for the old untreated rats (cf. Fig. 2). Similarly, in the young capsaicin-treated group, the vascular response is clearly decreased relative to the control (cf. Fig. 1A), but the abundances of mtDNA4834 are in the same range observed for the young untreated rats (data not shown in Fig. 4, but cf. Fig. 2). These results indicate that capsaicin treatment has an effect on vascular response superimposed on the deficits in this physiological function observed in normal senescent rats. However, the capsaicin-generated loss of vascular response appears to be independent of the accumulation of mtDNA mutations.

4. Discussion 4.1. Sensory nerve functional decline in ageing The decline of sensory nerve function in ageing is well documented both in rats and humans [20,21]. In humans this has been assessed, for example, by measurement of the so-called flare response following topical application of capsaicin onto the skin [13]. The magnitude of the flare response declines with age. Such responses to noxious stimulation of the skin are modulated by polymodal nociceptors of C fiber primary afferent neurons [13]. The same types of

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neurons are involved in the present studies, as the vascular response recorded in the rat is mediated via antidromic activation of C fibers in the sciatic nerve. The current study shows a clear effect of age on the physiological (vascular response) and biochemical (SP levels in DRGs) parameters reflecting a decline in C fiber function with age. Capsaicin treatment of neonates led to substantial ablation of these fibers during subsequent development that diminished both the vascular response and SP levels in DRG, in agreement with our previous findings [19,21]; this effect is not related to normal ageing. We now show here that the effect of age is superimposed on the capsaicin effect, such that in both untreated and capsaicin-treated animals vascular response and SP levels were significantly reduced in old compared to young rats. The parallel declines in SP levels in DRG and vascular response with age are consistent with the view that neuropeptides such as SP, released from peripheral sensory nerve are responsible for the observed vasodilatatory effects [14,15]. The sensory unmyelinated C fibers of the sciatic nerve are derived from particular DRG in the spinal lumbar region (primarily L3–L6) that act as source of SP for such neurons of sciatic nerve. For technical reasons DRG were collected from lower thoracic and all lumbar ganglia then pooled, ensuring availability of sufficient biological material for SP assays as well as for mtDNA mutational analyses (see below). The SP levels measured here are thus a composite of all such levels across the DRG population, but there is no reason to suspect that DRG from particular vertebral regions undergo substantially different losses of SP with age.

rats is of the order of 0.02% of total mtDNA. This is about 10 times greater than levels of mtDNA4834 reported for brain in comparably aged rats [7,49]. Whilst this may suggest that DRG accumulated mtDNA4834 at levels greater than those in brain, it is not clear if differences in the strains of rat used or in methods of PCR quantification contribute to this apparent differential abundance of mtDNA4834 in the peripheral, as opposed to central nervous system. Taking our data at face value, it seems that mtDNA4834 accumulates in DRG to similar extents to that seen in skeletal muscle of old rats [9]. What is striking in the present work is the prevalence of multiple deletions in mtDNA of DRG in old rats. Van Tuyle et al. [44] observed a wide range of abundant multiple deletions in mtDNA of rat brain. This may suggest a proclivity for neuronal tissue to accumulate multiple deletions during aging that is not exhibited by some other rat tissues including the skeletal muscle and heart [51]. Multiple deletions in human skeletal muscle and heart were readily observed by PCR techniques similar to those used here [28], in approximate proportion to the abundance of mtDNA4977. Brain of aging human subjects shows high levels of mtDNA4977, depending on the particular region analyzed [5,42], but data on mtDNA4977 in peripheral human neurons is not extensive. Detailed studies on multiple deletions in human brain or peripheral neurons have not, to our knowledge, been carried out by techniques similar to those reported here. In tissues of aging mice, application of longrange LX-PCR techniques [31], as well as conventional PCR [3,43] provided evidence of multiple deletions in brain.

4.2. Deletions in mtDNA of DRG

4.3. Relationship between loss of mtDNA integrity and decline in tissue physiological function

There is clear evidence that the particular deletion mtDNA4834 accumulates with age in DRG of the rat, to about a 300-fold extent comparing old and young rats. Such accumulation of mtDNA4834 is not affected by capsaicin treatment neonatally. Significantly, we have observed in DRG of old rats a very wide range of different mtDNA deletions detected by PCR, using primers differentially spaced on the target mtDNA genomes. Simply shifting one primer outwards by about 500 bases relative to the positions of those used in the primary detection and quantification of mtDNA4834 enabled visualisation of PCR products of typically 20 or more mtDNA deletions other than mtDNA4834 (estimated deletion size range 3.7 to 5.5 kb). Such deletions were not detected in DRG of young rats. Further spectra of mtDNA deletions in DRG of old (but not young) rats were observed using the widely spaced primer pair, in this case indicative of larger deletions in the range 5.2 to 7.5 kb in size. Strikingly, the patterns of PCR products arising from multiple deletions are unique to DRG samples from each individual rat. This suggests accumulation of multiple mtDNA deletions in DRG is extensive and stochastic. The abundance of mtDNA4834 in DRG of 24-month old

There is a striking correlation between mtDNA mutation level (indicated by quantified abundance of mtDNA4834 in DRG) and the loss of physiological function (represented by quantified loss of vascular function). Such a tight relationship has been observed in old untreated rats, and in both old and young capsaicin-treated rats. One can invoke two possible links between these tightly correlative parameters, reflecting (a) a functional or mechanistic link between the two observed parameters (direct process), or (b) an indirect link mediated by a third variable on which the observed two depend, but independently of one another (parallel process). The possibility of a direct link between mtDNA mutational levels, of which mtDNA4834 provides a convenient quantifiable index mutation, and vascular response could arise according to the following scenario. The accumulation of mitochondrial mutations in ageing could lead to debilitation of oxidative phosphorylation [26,35,38,39,45,47] that could reduce ATP production in DRG. This in turn could limit the biosynthesis of SP and other sensory neuropeptides, leading to their depletion from DRG (as observed in the case of SP), with consequent loss of vascular function

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(also observed). Other more subtle or complex mechanistic interactions could be envisaged, more or less along the same route leading directly from mtDNA mutation to loss of physiological function. Such scenarios do not imply a particular mechanism for induction of mtDNA mutations, but a commonly invoked cause is oxidative stress [35,38,47]. Nevertheless, the global abundance of mtDNA mutations in DRG of old rats may be insufficient, in itself, to cause loss of mitochondrial function. An alternative explanation for the data may occur as a result of parallel processes leading to mtDNA mutation, on the one hand, and loss of physiological function, on the other. One primary cause of both these outcomes could be oxidative stress. Oxidative damage could lead to accumulating damage to proteins, lipids and biological membranes that directly reduces tissue functions [reviewed in 11]. One view is that the mtDNA damage observed merely represents the molecular imprint of the oxidative stress on mtDNA. It is moreover argued that such mtDNA damage occurs to such a small fraction of molecules in the cellular pools of mtDNA that mitochondrial function could not be significantly affected [25]. Nevertheless, others attribute negative mitochondrial and cellular functional consequences to the intracellular population of mtDNA mutations that are represented at relatively low levels individually, but which collectively combine to impair cumulatively mitochondrial (and consequently, cellular) functions [35]. It is entirely possible that there is a combination of direct and parallel processes that leads to the observed tight correlation between mtDNA4834 levels and loss of vascular function. The correlative abundance of mtDNA4834 and loss of function may not apply to all tissues in the rat, exemplified by the liver). In hepatocytes of rats of various ages the mitochondrial membrane potential was observed to decline with age, but this was not accompanied by a corresponding increase in the abundance of mtDNA4834 [10].

Acknowledgment This work was supported by grants to Z.K. from the National Health and Medical Research Council of Australia.

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