Brain, Vol. 122, No. 11, 2109-2118,
November 1999
© 1999 Oxford University Press
Invited review |
Neurofilament and tubulin gene expression in progressive experimental diabetes
Failure of synthesis and export by sensory neurons
1 Department of Clinical Neurosciences and Neuroscience Research Group and 2 Departments of Pathology and Clinical Neurosciences, and Neuroscience Research Group, The University of Calgary
Correspondence to:
Dr D. W. Zochodne, University of Calgary, 182a Heritage Medical Research Building, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1 E-mail: dzochodn{at}ucalgary.ca
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Abstract |
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In human and experimental diabetes, the relationship between molecular abnormalities in perikarya of sensory neurons and structural abnormalities in their distal axons is largely unexplored. In this study we examined neurofilament (Nf) and tubulin messenger RNA (mRNA) expression and their incorporation into distal sensory axons during progressive streptozotocin-induced diabetes in rats. After 2 and 6 months of diabetes, we measured mRNA levels of all three Nf subunits, B50 [growth associated protein-43 (GAP-43)] and
-tubulin in L4–L6 dorsal root ganglia using Northern analysis. The same animals underwent morphometric studies of myelinated fibres by light microscopy and quantitative analysis of Nf and microtubule numbers and density within sural myelinated and unmyelinated axons. Multifibre in vivo sensory and motor conduction nerve recordings confirmed slowing of conduction velocities in diabetic rats indicating experimental neuropathy. mRNA levels for the three Nf subunits, B50 (GAP-43) and -tubulin were unchanged from controls at 2 months, but were decreased by 26–46% at 6 months. These changes accompanied declines in Nf numbers and densities within large myelinated sensory axons, and Nf numbers in unmyelinated fibres by 6 months. Microtubule numbers and densities were similarly reduced in large myelinated axons, and microtubule numbers reduced in small myelinated and unmyelinated axons in diabetes at 6, but not 2 months. Axonal atrophy was observed in unmyelinated fibres at 6 months. Our findings indicate that decreased mRNA expression of cytoskeletal proteins in sensory neurons accompanies a reduction in their incorporation into distal axons. These changes imply that there is a direct link between pathological changes in the sensory neuron and alterations of its distal branches from experimental diabetes. The changes in gene expression in diabetes are unique and differ from those that develop after axotomy. diabetic neuropathy; dorsal root ganglia; conduction velocity; streptozotocin
GAP-43 = growth associated protein-43 (B50); mRNA = messenger RNA; Nf = neurofilament; Nf-H = Nf-heavy subunit; Nf-L = Nf-light subunit; Nf-M = Nf-medium subunit; STZ = streptozotocin
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Introduction |
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A distal sensory polyneuropathy is the most common form of peripheral nerve disorder in diabetic subjects. The role of sensory ganglia dysfunction in this condition is largely unexplored and it is uncertain whether abnormalities at the perikaryal level might predict structural abnormalities in distal axons (Zochodne, 1996a
). Neurofilaments (Nfs) are major determinants of axonal calibre, particularly of large myelinated axons. Correlations between Nf gene expression, axonal calibre and axonal Nf content occur during development, maturation, regeneration and ageing (Hoffman et al., 1985, 1987; Muma et al., 1990, 1991; Scott et al., 1991; Tetzlaff et al., 1991; Parhad et al., 1995). Axonal Nf deficiency could result from a defective perikaryal synthesis, abnormal transport or an increased turnover in the axon. Recently, Mohiuddin and colleagues (Mohiuddin et al., 1995) suggested that experimental diabetes was associated with a decline in the expression of two of the three Nf subunits in sensory neurons. However, it is not known whether such alterations in Nf gene expression or those of tubulin correlate with abnormalities in conduction velocity, myelinated fibre calibre or incorporation of Nfs and microtubules into distal axons.
In the present study, we examined whether early or longer-term streptozotocin (STZ)-induced diabetes was associated with progressive alterations of Nf and -tubulin messenger RNA (mRNA) expression in lumbar sensory ganglia. The results of neuronal gene expression were then compared with quantitative studies of fibre calibre, and of sural axonal Nf and microtubule incorporation. The findings suggested an important relationship.
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Material and methods |
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Experimental design
The experimental protocol was reviewed and approved by the University of Calgary Animal Care Committee. Male Sprague–Dawley rats of initial weight 230–300 g (age ~12 weeks) were randomly divided and injected with either a single dose of i.p. (intraperitoneal) STZ (Zanosar, Upjohn; 65 mg/kg) in citrate buffer, or an equal volume of citrate buffer alone. Blood glucose measurements were made 5–7 days after injection and the rats were considered diabetic only if the values were
12 mmol/l throughout the studies (measured at 7 days, 2 months and 6 months following injection). Glucose measurements were made from the ventral caudal vein using a glucometer (Accuchek II; Boehringer–Mannheim, Laval, Quebec, Canada). The rats were housed in grouped plastic-bottomed cages with free access to rat chow and water. Rats were sacrificed at 2 and 6 months with pentobarbital (130 mg/kg, i.p.) and bilateral L4–L6 dorsal root ganglia were dissected, weighed and frozen at -80o C for Northern analysis. The sural nerves distal to the sciatic trifurcation were dissected for morphometric analysis.
Multifibre nerve conduction recordings
Multifibre in vivo nerve conduction recordings were made prior to the injection of STZ or buffer and repeated at 2 and 6 months. Recordings were made in anaesthetized rats (pentobarbital 65 mg/kg, i.p.) and included measurements of sensory caudal (the nerve is mixed motor and sensory but conduction velocity is determined by the faster conducting sensory fibres) and sciatic-tibial motor conduction (Zochodne and Ho, 1992). Subcutaneous near nerve temperatures were maintained at 37 ± 1°C during the conduction measurements. Latencies were measured to the onset of the negative deflection of the potential, and amplitudes calculated from baseline to peak.
Northern analysis
Total RNA was isolated from L4–L6 dorsal root ganglia using the acid guanidinium thiocyanate–phenol–chloroform extraction technique (Chomczynski and Sacchi, 1987). Gel fractionation, transfer to nylon membranes and repeated Northern hybridization were performed as previously described (Parhad et al., 1995). Each membrane contained samples from several animals of both experimental groups at each time point. Hybridizations were performed at high stringency [hybridized at 60°C and washed at 65–70°C with 0.1 x SSC (standard saline citrate); 1 x SSC = 0.15 M sodium chloride and 0.15 M sodium citrate] and the membranes were exposed to Kodak X-ray film for 16–48 h. The density and area of specific bands on autoradiograms were quantified by video densitometry (Bioquant System IV; R & M Biometrics Inc., Nashville, Tenn., USA) as previously described (Parhad et al., 1992). The ethidium bromide-stained 28S and 18S bands on the filters were similarly quantified, and the sum of the two bands was used as the denominator for the Northern blots. For each diabetic mRNA, the data were normalized to the value of the control non-diabetic dorsal root ganglia sample.
Selection of cDNA probes
Probes were prepared from the following clones: a 2-kb mouse Nf-light subunit (Nf-L) cDNA, a 660-bp mouse Nf-medium subunit (Nf-M) cDNA and a genomic BglII fragment of the mouse Nf-heavy subunit (Nf-H) (Julien et al., 1986); a 1.1-kb rat B50 (GAP-43) cDNA encompassing the full coding sequence (Basi et al., 1987); and a 192 bp MboII fragment of mouse 1-tubulin cDNA consisting exclusively of the 3'-untranslated region. This last region is specific for 1-tubulin and shows extensive homology among species. The DNA clones were amplified and purified by standard techniques (Sambrook et al., 1989) and labelled with [-32P]dCTP for Northern analysis (Du Pont Canada Inc., Mississauga, Ontario, Canada) using a random prime labelling kit (Boehringer–Mannheim) to specific activities >1 x 109 c.p.m./µg.
Morphometric analysis
Sural nerves from diabetic and non-diabetic rats 2 and 6 months after STZ and citrate injection, respectively, were harvested for light and electron microscopic analysis. At the sampled level the nerves were largely monofascicular. The nerves were immersion fixed in glutaraldehyde (2.5%) buffered with cacodylate (0.025 M) overnight, dehydrated in graded alcohols, stained with osmium tetroxide and embedded in epon. Epon sections of 1 µm were stained with toluidine blue, and myelinated fibres were counted and sized using a video-computer image analysis program (Auer, 1994). Electron micrographs with a final magnification x24 000 (large myelinated axons) and x40 800 (small myelinated and unmyelinated axons) were used for the measurement of axonal size and analysis of the axonal cytoskeleton. Randomly selected myelinated and unmyelinated fibres from each nerve fascicle were measured. At least six large (6 µm diameter) and six small (
6 µm diameter) myelinated axons and 50 unmyelinated axons were evaluated for each of three diabetic and control animals. Nf counting was carried out by an observer blinded to the treatment group. For the large myelinated fibres, we chose the largest fibres observed that were technically suitable for electron microscopic counting. The axonal area was measured using a video-computer image analysis program (Auer, 1994). Nfs and microtubules cut perpendicularly were counted within the total axonal area of each fibre. For each rat mean values were calculated from the above and the means and standard errors then calculated for each group.
Statistical analysis
The data were expressed as mean ± standard error of the mean. For comparison of two means, the data were analysed with either a two-sample (unpaired) Student's t-test or non-parametric (Mann–Whitney) test. For electron microscopy, statistical analysis was based on n = number of rats studied.
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Results |
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Diabetic and electrophysiological assessment
Diabetic rats had hyperglycaemia and gained less weight than non-diabetic rats over both study periods. Results of the final weights and glucose levels are given in Table 1
. Multifibre in vivo sensory and motor nerve conduction recordings confirmed slowing of conduction velocities in the diabetic animals in the caudal sensory and sciatic–tibial motor fibres at both 2 and 6 months, compared with controls (Fig. 1).
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Nf mRNA levels in diabetic dorsal root ganglia neurons
mRNA levels for Nf-L, Nf-M and Nf-H in diabetic animals were unchanged from controls at 2 months. Similarly, B50 (GAP-43) and
-tubulin were comparable with controls at 2 months. At 6 months, however, all three Nf subunit mRNA levels had significantly declined by between 26 and 46%. Comparable declines in B50 (GAP-43) and -tubulin mRNA levels were observed. Results are given in Table 2.
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Light microscopic morphometry
Results of the light microscopic morphometry studies are given in Table 3
and myelinated fibre size histograms in Fig. 2. At 2 months there was a slight but non-statistical shift towards fewer, larger fibres and increased numbers of smaller myelinated fibres in the diabetic rats, with a borderline reduction of mean fibre diameter suggesting mild axonal atrophy (Table 3). However, axonal diameter, fibre area and axonal areas were comparable between the diabetic and non-diabetic rats. At 6 months there was no definite evidence of fibre atrophy using light microscopy despite a borderline significant trend shown with the electron microscopy studies (see below). There was a non-significant trend towards decreased numbers of myelinated fibres and myelin thinning in diabetic rats at 6 months.
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Electron microscopic morphometry of the axonal cytoskeleton
Comparison of the axonal cytoskeleton for diabetic and control rats is summarized in Table 4
. In non-diabetic rats there was an increase in the cross-sectional axonal area and a trend towards increasing numbers of Nfs, with decreased Nf density between 2 and 6 months. In contrast, small myelinated fibres and unmyelinated fibres had similar Nf content at the two time points. Microtubule numbers increased between 2 and 6 months in non-diabetic rats.
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There was no difference in the number of axonal Nfs or Nf density between diabetic and control rats at 2 months. At 6 months there was a reduction in Nf numbers and Nf density in large myelinated fibres of diabetic rats, compared with controls. In small myelinated fibres there were non-significant trends toward fewer numbers of Nfs and axonal atrophy. Nf numbers, but not density, were reduced in unmyelinated fibres and there was axonal atrophy. Microtubule numbers and densities were not reduced in large or small myelinated fibres, or unmyelinated fibres at 2 months. At 6 months there were declines in microtubule numbers and density in large myelinated fibres and a decline in microtubule numbers in small myelinated fibres and unmyelinated fibres (Figs 3 and 4
).
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Discussion |
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The major findings from this work were: (i) early (2 month) and later (6 month) experimental diabetes were associated with reductions in motor and sensory conduction velocities; (ii) there was a substantial decline in the expression of all three Nf subunits, of
-tubulin and of B50 (GAP-43) by 6, but not by 2 months of diabetes; (iii) comparable declines in the diabetic rats at 6, but not 2 months were observed in the incorporation of Nf and microtubules into axons, most noticeably in large myelinated fibres, but this was not associated with atrophy. Borderline atrophy was observed in small myelinated fibres and definite atrophy was seen in unmyelinated fibres; (iv) changes in gene expression predicted those of axonal structure; (v) the pattern of progressive impairment of gene expression differed substantially from that expected from axotomy.
Nfs are the major intermediate (10 nm) filaments in many types of mature neurons. Assembled as obligate heteropolymers of three polypeptide subunits, Nf-L, Nf-M and Nf-H, Nfs are the most abundant cytoskeletal component in large myelinated axons, particularly those arising from large lower and upper motor and sensory neurons. The quantity of Nf proteins within an axonal segment is determined by the rates of Nf synthesis, transport, phosphorylation and degradation. During development and maturation there is an increase in Nf mRNA levels, a decrease in the rate of Nf transport and an increase in Nf-H phosphorylation, each of which contributes to the increased quantity of Nf proteins within the axon (Carden et al., 1987; Dahl, 1988; McQuarrie et al., 1989; Muma et al., 1991). Following axotomy, a decrease in Nf gene expression appears to predict declines in axon calibre (Hoffman et al., 1987). Previous reports have suggested that changes of axonal calibre in STZ-induced diabetic rats are secondary to reductions in axonal Nf and microtubule content, in turn caused by diabetes-related impairment of slow axonal transport (Medori et al., 1988a, b; Macioce et al., 1989). Our study suggests instead that decreased Nf and -tubulin mRNA expression, and an impaired Nf and tubulin supply directly predict declines in the axonal incorporation of Nfs and microtubules. Altered axonal insertion was most obvious in large myelinated fibres and unmyelinated fibres. A similar trend was observed in smaller myelinated fibres, but was not statistically significant, possibly because there are fewer Nfs to sample in this population. The relationship of this incorporation to axonal atrophy, however, was less robust in myelinated fibres. We did not observe significant atrophy except in unmyelinated fibres at 6 months. Since many more fibres are sampled using light microscopy, we relied on this technique to draw our final conclusions about myelinated fibre calibre. It is possible that changes in structural proteins, however, are associated with more distal sensory terminal atrophy or retraction that we did not specifically seek. Yagihashi and colleagues also observed a reduction in Nf content of sural myelinated fibres that exceeded the loss predicted by axon size (Yagihashi et al., 1990). In their model, axonal atrophy was more prominent in myelinated fibres after 7 months of diabetes, and they postulated that reductions in neuronal synthesis of Nfs accounted for their findings.
Our results probably lend support to those of Mohiuddin and colleagues who suggested that expression of two of the three Nf subunit mRNAs declines in short-term (2 months) experimental diabetes (Mohiuddin et al., 1995). While we did not observe changes by 2 months, differences in rat strain susceptibility and diabetes intensity could account for the time differences. We confirmed, however, that Nf synthesis does decline by 6 months and that it is substantial and involves all three Nf subunits. Such a decline could reflect fewer neurons producing Nf, but we did not observe a statistically significant decline in sural fibre numbers despite a trend in this direction. Accurate neuronal counts without bias using a dissector approach and in a long-term study of diabetes have not been published. We also think it is unlikely that Nf mRNA was somehow `diluted' by a large increase in the non-neuronal cell population. Moreover, the mRNA findings matched the electron microscopy counts of Nf protein. Separate work in 12-month diabetic rats has identified Nf mRNA reductions using quantitative in situ hybridization (D. W. Zochodne and V. M. Verge, unpublished data). Finally, unlike previous work, we also compared the changes in gene expression with alterations in conduction velocity. There was no relationship.
The most consistent morphometric finding reported in animals rendered diabetic before the cessation of their growth period has been a slight reduction in myelinated fibre size in diabetic animals compared with age-matched controls (Sharma et al., 1981; Mattingly and Fischer, 1985; Thomas et al., 1990). This change has been postulated to be due to a retardation in normal growth rates secondary to diabetes. However, other studies have described a proximal increase and distal decrease in axonal calibre in STZ-induced neuropathy that is associated with changes in axonal Nf number and explained by impaired anterograde axonal transport (Medori et al., 1988a). The slower axonal transport of Nfs is thought to lead to a thickening of the axons in the proximal segments and to a reduction of axonal calibre in the distal parts. A complicating factor is that most studies evaluating the effects of experimental diabetes on nerve fibre size and Nf content have included rats that had not yet reached full maturity (Medori et al., 1988a; Mohiuddin et al., 1995). Some investigators have questioned whether smaller axonal size is simply due to a delay in maturation rather than active axonal atrophy (Thomas et al., 1990; Weis et al., 1995). In contrast, other reports using more mature or older diabetics (Wright and Nukada, 1994), as in our 6-month work, have found no significant differences in axonal calibre between control and diabetic groups. Unmyelinated fibres have not been studied. As in previous work using this model, a lesser weight gain was observed in diabetic rats compared with controls. While this raises a concern that changes observed may reflect a nutritional deficiency, we observed altered gene expression at the 6-month mark, whereas differences in weight gain were much more pronounced at 2 months when gene expression was unaltered. Our choice of a 6-month time point was a deliberate approach to this problem.
Overall, our findings suggest that in experimental diabetes of sufficient duration there are less than direct connections between changes in conduction velocity, axonal calibre and axonal incorporation of structural proteins. Each may change in parallel during diabetes but for separate reasons at different times. It may be that declines in gene expression of structural proteins will become a better predictor of distal sensory terminal retraction and loss, the most important feature of clinical sensory loss in diabetes. Conduction velocity changes in diabetes are probably better accounted for by metabolic changes, such as sodium accumulation in axons or sodium channel migration out of nodes of Ranvier (Greene et al., 1987; Cherian et al., 1996).
Axotomy of peripheral nerves normally provokes changes in synthesis of neuronal proteins thought to be necessary for axonal regeneration. Sciatic nerve section causes a marked decrease in Nf, and marked increases in B50 (GAP-43) and tubulin mRNA levels in the lumbar dorsal root ganglia (Scott et al., 1991; Scott and Parhad, 1994; Verge et al., 1996), even in the diabetic state (Pekiner et al., 1996). Nerve growth factor can rescue these changes (Gold et al., 1991; Verge et al., 1996; Zochodne, 1996b). Axotomy also provokes a significant increase in the anterograde axonal transport of B50 (GAP-43) in diabetes (Tetzlaff et al., 1989), and the established correlation between its increased synthesis and transport suggests that B50 (GAP-43) participates in regeneration. In the present study and others (Mohiuddin et al., 1995; Pekiner et al., 1996) the decrease in mRNA for B50 (GAP-43) could well be a factor contributing to the impaired regeneration characteristic for this model. Thus, taken together, the data on Nf and B50 (GAP-43) mRNAs indicate an impairment in the steady-state expression of cytoskeletal and growth-associated proteins in lumbar dorsal root ganglia of diabetic rats. The alterations we observed, however, are unique and clearly differ from those after axotomy and may argue against NGF deficiency as the sole mechanism for the development of diabetic neuropathy. It is possible that impaired neurotrophin-like actions of insulin, insulin-like growth factor-1 or other factors instead account for the unique alterations in sensory neuron gene expression.
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Acknowledgments |
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We wish to thank Hong Sun, Chu Cheng and Sonny Bou for technical assistance, Brenda Boake for expert secretarial assistance and Dr J.-P. Julien for providing cDNA probes (Nf-L, Nf-H and Nf-M). This work was supported by grant funding from the Medical Research Council of Canada. D.W.Z. is a Medical Scholar of the Alberta Heritage Foundation for Medical Research. This paper was presented in abstract form at the 1996 American Neurologic Association Meeting (Scott JN, Clark AW, Chapman K, Sun H, Bou S, Zochodne D. Neurofilament gene expression in experimental diabetes. Ann Neurol 1996; 40: 514).
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Received March 29, 1999. Accepted May 24, 1999.
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N. M. Sayers, L. J. Beswick, A. Middlemas, N. A. Calcutt, A. P. Mizisin, D. R. Tomlinson, and P. Fernyhough Neurotrophin-3 Prevents the Proximal Accumulation of Neurofilament Proteins in Sensory Neurons of Streptozocin-Induced Diabetic Rats Diabetes, September 1, 2003; 52(9): 2372 - 2380. [Abstract] [Full Text] [PDF]
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M. Kishi, J. Tanabe, J. D. Schmelzer, and P. A. Low Morphometry of Dorsal Root Ganglion in Chronic Experimental Diabetic Neuropathy Diabetes, March 1, 2002; 51(3): 819 - 824. [Abstract] [Full Text] [PDF]
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S. Yagihashi, S.-I. Yamagishi, R.-i. Wada, M. Baba, T. C. Hohman, C. Yabe-Nishimura, and Y. Kokai Neuropathy in diabetic mice overexpressing human aldose reductase and effects of aldose reductase inhibitor Brain, December 1, 2001; 124(12): 2448 - 2458. [Abstract] [Full Text] [PDF]
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D. W. Zochodne, V. M. K. Verge, C. Cheng, H. Sun, and J. Johnston Does diabetes target ganglion neurones?: Progressive sensory neurone involvement in long-term experimental diabetes Brain, November 1, 2001; 124(11): 2319 - 2334. [Abstract] [Full Text] [PDF]
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 Y. Shimoni and J. B. Rattner Type 1 diabetes leads to cytoskeleton changes that are reflected in insulin action on rat cardiac K+ currents Am J Physiol Endocrinol Metab, September 1, 2001; 281(3): E575 - E585. [Abstract] [Full Text] [PDF]
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J. M. Kennedy and D. W. Zochodne The regenerative deficit of peripheral nerves in experimental diabetes: its extent, timing and possible mechanisms Brain, October 1, 2000; 123(10): 2118 - 2129. [Abstract] [Full Text] [PDF]
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