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Immunocytochemical studies of neurotrophins in cerebral motor cortex in Amyotrophic Lateral Sclerosis
Brain Research 763 Ž1997. 259–263
Short communication
Immunocytochemical studies of neurotrophins in cerebral motor cortex in Amyotrophic Lateral Sclerosis R.M. Duberley a
a,b
, I.P. Johnson
a, )
, P. Anand b , P.N. Leigh c , N.J. Cairns
d
Department of Anatomy and DeÕelopmental Biology, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, UK Department of Neurology, Saint Bartholomew’s and The Royal London Hospital School of Medicine and Dentistry, London E1 lBB, UK c Department of Clinical Neuroscience, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK d Department of Neuropathology, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK
b
Accepted 1 April 1997
Abstract Neurotrophin-like immunoreactivity was studied in post-mortem motor cerebral cortex from patients with Amyotrophic Lateral Sclerosis ŽALS. and controls. Neurotrophin-4r5 immunoreactivity was seen in small-Ž12–25 mm., medium-Ž26–39 mm., and large-Ž) 40 mm., neurones, neurotrophin-3 was seen in medium and small neurones, while brain-derived neurotrophic factor was restricted to small neurones. No difference in number or intensity of immunostained neurones was found between ALS and controls. q 1997 Elsevier Science B.V. Keywords: Amyotrophic lateral sclerosis; Motor cortex; Neurotrophic factor; Immunocytochemistry
Amyotrophic lateral sclerosis ŽALS. is a neurodegenerative disorder characterised by progressive muscle weakness, combined with symptoms of corticospinal tract degeneration w27x. Motoneurones in the ventral horn of the spinal cord are lost, as are brainstem motoneurones and pyramidal neurones of the cerebral motor cortex; the number of motoneurones lost in these regions varies with the clinical sub-type of the disease w15,18x. The aetiology of the most common form of this disease Žsporadic ALS. remains unknown. Nevertheless, hopes for a therapy have been raised by recent animal studies which have demonstrated the survival-promoting properties of neurotrophic factors on spinal motoneurones w3,11,12,24,25x, prompting clinical trials of systemically-administered neurotrophic factors, such as brain-derived neurotrophic factor ŽBDNF. and insulin-like growth factor-1 ŽIGF-1. and ciliary neurotrophic factor ŽCNTF.. We found previously that levels of CNTF, but not nerve growth factor ŽNGF., were decreased in the ventral horn of ALS spinal cord w2,6x. More recently, we found that ALS spinal motoneurones had reduced neurotrophin-3 ŽNT-3.-like immunoreactivity compared to controls w7x. Taken together with reports of
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changes in the receptors for IGF- 1, BDNF and CNTF in spinal motoneurones in ALS w1,6,23x, these observations serve to emphasise the potential importance of neurotrophic factors in ALS. All these studies are, however, concerned with the spinal cord and lower motoneurones, and the extent to which neurotrophic factors or their receptors are involved in upper motoneuronal loss remains unclear. We have previously found that mRNA levels of both CNTF the a subunit of the CNTF receptor in the motor cerebral cortex are unchanged in ALS w2,6x, with very low levels of CNTF measured by ELISA in motor and occipital cerebral cortex in comparison with the spinal cord. Interestingly, NGF levels are decreased in ALS motor cortex, but not occipital cortex w2x, and increased levels are found in the dorsolateral fasciculus of the spinal cord where degenerating corticospinal fibres are found. Little is known, however, of the expression of other neurotrophins in ALS. In this study, therefore, we have used immunocytochemical methods to study the neurotrophins NT-3, neurotrophin 4r5 ŽNT-4r5. and BDNF in postmortem human motor cortices of control and ALS patients. Motor cerebral cortex was obtained post-mortem from eight patients aged 58–71 years, clinically diagnosed with sporadic ALS, and 5 age-matched control patients Ž63–83 years. with no clinical history of neurological disease. The
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mean post-mortem interval for ALS patients was 18.8 h Žrange 4–24 h. and for control patients was 23.4 h Žrange 19–26 h.. Tissue was snap-frozen on a brass plate at y708C and stored at y708C until required. The presence of large Ž) 40 mm. pyramidal neurones in cortical layer V of cryostat sections stained with 0.05% Cresyl fast violet was taken as confirmation that the sections were from the motor cortex region. As expected, fewer Betz cells were found in ALS tissue. After confirming the identity of the
tissue block, cryostat sections Ž20 mm. were mounted on chrome-alum-subbed slides, allowed to equilibrate to room temperature, then fixed for 30 min in 4% paraformaldehyde in phosphate buffered saline ŽPBS., pH 7.4. After rinsing twice in 0.1 M PBS, pH 7.4, slides were incubated in 0.2% Triton X-100 in PBS for 1 h to improve the access of antibodies to tissue antigens. Slides were then washed in PBS and incubated with 0.05% H 2 0 2 in PBS for 20 min to block endogenous peroxidase activity. Slides were again
Fig. 1. NT-3, NT-4 and BDNF-like immunoreactivity in the motor cortex of control and ALS patients. Scale bar s 40 mm.
R.M. Duberley et al.r Brain Research 763 (1997) 259–263
washed in PBS prior to a 20 min incubation with normal goat or horse serum diluted 1:30 in PBS to reduce background immunostaining. Sections were blotted to remove excess blocking serum and then incubated with the primary antibody in PBS containing 0.03% Triton X-100, 0.1% sodium azide and 0.1% bovine serum albumin ŽBSA. for 24 h at 48C. The BDNF antibody was raised in rabbits against E. coli-derived recombinant human BDNF and shown not to cross-react with nerve growth factor, NT-3 or NT-4 w34x. Similar, but unpublished characterisation has been carried out for the NT-3 Žpers. commun., J. Carnaham, Amgen. and NT-4r5 antibodies. The NT-4r5 staining was also confirmed using a another, commerciallyavailable, polyclonal antibody ŽSanta Cruz Biotechnology, Inc., USA, dilution: 1:1K. raised to a 20 amino acid peptide sequence of the mature form of human NT-4r5 Ždata not shown.. The working dilutions, source and reference numbers of the primary antibodies used in this study are summarised below: Polyclonal BDNF Ž1:5K. ŽAmgen Inc. USA., ŽRef. 7253-34 ŽA..; Polyclonal NT-3 Ž1:lOK. ŽAmgen Inc. USA., ŽRef. 8Cr845 a883.; Polyclonal NT-4r5 Ž1:lK. ŽDr. D. Kaplan, Advanced BioScience Labs.., ŽRef. a NT-4. After incubation with the primary antibody, sections were rinsed in 0.025% Tween 20rPBS, washed in PBS and then incubated in the biotinylated secondary antibody at a dilution of 1:100 in PBS for 1 h. Following a further rinse in 0.025% Tween 20rPBS and three PBS washes, the sections were incubated for 1 h in an avidin-biotin ŽABC. complex Ž1: 100 in PBS containing 0.1% BSA without azide; Vector Labs... Slides were again rinsed in 0.025% Tween 20rPBS, followed by three washes in PBS. Staining was then visualised by incubating the slides with 0.05% diaminobenzidine containing 2.5% nickel sulphate and 0.001% glucose oxidase w28x for 5 to 15 min. Sections were then dehydrated and mounted in DPX. Immunostaining was abolished by overnight preabsorption of the antibody at 48C with 100 ng-200 mgrml of the corresponding antigen Žrecombinant human BDNF, NT-3 or NT-4r5, Amgen.. Omission of primary or secondary antibodies produced no staining, while preabsorption of the
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antibody with inappropriate antigens had no effect on immunostaining. Using a =40 objective lens and a X10 eyepiece, the numbers and mean diameters Žmax q minr2. of immunostained and non-immunostained neurones in randomlyplaced fields of 855 mm2 area, over layer V of the motor cortex were determined without knowledge of the slide codes for 4–8 slides per subject. For each neurotrophin, the percentage of all neurones showing neurotrophin-like immunoreactivity was determined. NT-3-like immunoreactivity was detected in small Ž12– 25 mm. and medium Ž26–39 mm. diameter neurones within the motor cortex ŽFig. 1., but not in large neurones. No obvious differences of staining intensity or numbers of immunopositive cells in control Ž49.0 " 10.0% positive cells. and ALS patients Ž52.5 " 5.4% positive cells. were seen ŽTable 1.. NT-4r5-like immunoreactivity using both sources of primary antibody was present within small and medium-diameter neurones Žrange: 12–32 mm. in the motor cortex of control Ž51.0 " 10.2% positive cells. and ALS patients Ž49.1 " 11.9% positive cells.. Occasional staining of larger pyramidal neurones Ž49–55 mm diameter. of both control and ALS patients was also seen. Some small Ž- 10 mm. presumptive neuroglial cells within the motor cortex were also immunopositive for both NT-3 and NT-4r5. Occasional weak BDNF-like immunostaining of small neurones Ždiameter range: 12–28 mm. within the motor cortices of control Ž21.3 " 7.7% positive cells. and ALS patients Ž21.7 " 9.9% positive cells. was seen. Our immunocytochemical studies have shown that, in general, neurotrophic factors are localised to the cell bodies of small and medium-sized neurones of the cerebral motor cortex, and to presumptive neuroglia. Both neurones and neuroglia synthesise a variety of neurotrophic factors w13,14,20,26,29,30x and these can be taken up and transported retrogradely or anterogradely by neurons w16,31– 33x. In the absence of additional in situ hybridisation studies to define the cellular location of the mRNAs, however, we cannot estimate the extent to which our immunocytochemical findings reflect the synthesis of neurotrophic factors by the labelled cells or uptake from other sources. Such information could also help rationalise some apparent discrepancies between reported levels of neu-
Table 1 Mean number of neurones and the proportion showing neurotrophin-like immunoreactivity within area V of the motor cerebral cortex of five control and eight ALS patients Control
BDNF NT-3 NT-4
Mean S.D. Mean S.D. Mean S.D.
ALS
Total
Positive
% Positive
Total
Positive
% Positive
16.4 5.8 14.9 4.6 10.0 1.3
3.5 0.6 7.3 2.3 5.1 1.1
21.3 7.7 49.0 10.0 51.0 10.2
14.3 4.8 20.2 6.1 16.7 5.6
3.1 1.0 10.6 4.0 8.2 3.1
21.7 9.9 52.5 5.4 49.1 11.9
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rotrophic factors and their receptors. Thus, increased expression of trkB and p75 NG FR has been found for motoneurones in ALS spinal cord w23x, without increased NGF binding w9x or increased immunostaining of motoneurones for NGF or BDNF Žunpubl. obs... Large-diameter pyramidal neurones of the motor cortex have classically been equated with Betz cells, which contribute axons to the corticospinal tract. In contrast to large-diameter spinal motoneurones w6,22x, however, we found little staining of presumptive Betz cells for neurotrophic factors, and no obvious changes in ALS. This finding may be a reflection of the low number of Betz cells seen normally in single sections in the present study and the further sampling problems imposed by their loss in ALS. Alternatively, the absence of neurotrophic factor changes in Betz cells could be taken to indicate that mechanisms other than those involving neurotrophic factors contribute towards their loss. This proposal does not preclude the involvement of neurotrophic factors in the loss of medium and small neurones. It is likely that many of these smaller neurones contributed to the corticospinal tract, given that the Betz cell contribution may account for less that 5% of fibres w19x. Other immunolabelled cortical neurones observed in this study may have been local interneurones. Their persistence in the face of a reduction in the reference area due to the general atrophy of layer V may explain our finding that the density of neurones of all sizes was slightly increased in ALS cortex compared to controls ŽTable 1.. Animal studies have demonstrated the importance of neurotrophic factors in promoting the survival and sprouting of injured corticospinal neurones w5,21x, as well as the structural and functional development of the visual cortex w10,30x. As both situations involve modifications of the connectivity of cortical interneurones and projection neurones, it is possible that one role of neurotrophic factors in the cerebral motor cortex is to maintain synaptic connectivity. Such a role assumes the intraneuronal transport of neurotrophic factors, and it is interesting that defective axonal transport associated with overexpression of the human heavy neurofilament subunit gene has recently been shown in a transgenic mouse model of ALS w4x. In the context of theories on the pathogenesis of ALS w8,17x, it will be important, if suitable markers of interneurones and upper motoneurones can be found, to determine the relative proportion of corticospinal neurones to interneurones which are immunopositive for neurotrophic factors and the relative degree of neuronal degeneration occurring in these two populations.
Acknowledgements This work was supported by a grant from the Motor Neurone Disease Association, UK. The NT-4r5 antibody was a gift from Dr. D. Kaplan ŽAdvanced BioScience
Laboratories., the BDNF and NT-3 antibodies and neurotrophins were gifts from Amgen Inc. Equipment grants were received from the Medical Research Council ŽUK. and the Central Research Fund ŽUniversity of London.. References w1x A. Adem, J. Ekblom, P.G. Gillberg, S.S. Jossan, A. Hoog, B. Winblad, S.M. Aquilonius, L.H. Wang, V. Sara, Insulin-like growth factor-1 receptors in human spinal cord: changes in amyotrophic lateral sclerosis, J. Neural Transm. 97 Ž1994. 73–84. w2x P. Anand, A. Parrett, J. Martin, S. Zeman, P. Foley, M. Swash, P.N. Leigh, J.M. Cedarbaum, R.M. Lindsay, R.E. Williams-Chestnut, D.V. Sinicropi, Regional changes of ciliary neurotrophic factor and nerve growth factor levels in post mortem spinal cord and cerebral cortex from patients with motor disease, Nature Med. 1 Ž1995. 168–172. w3x Y. Arakawa, M. Sendtner, H. Thoenen, Survival effect of ciliary neurotrophic factor ŽCNTF. on chick embryonic motoneurons in culture: comparison with other neurotrophic factors and cytokines, J. Neurosci. 10 Ž1990. 3507–3515. w4x J.F. Collard, F. Cote, J.P. Julien, Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis, Nature 375 Ž1995. 61–64. w5x S.M. Dale, R.Z. Kuang, X. Wei, S. Varon, Corticospinal motoneurons in the adult rat: degeneration after intracortical axotomy and protection by ciliary neurotrophic factor ŽCNTF., Exp. Neurol. 135 Ž1995. 67–73. w6x R.M. Duberley, I.P. Johnson, M. Swash, J. Martin, P.N. Leigh, S. Zeman, Ciliary neurotrophic factor expression in spinal cord and motor cortex in amyotrophic lateral sclerosis, J. Neurol. Sci. ŽSuppl.. 129 Ž1995. 109–113. w7x R.M. Duberley, I.P. Johnson, P. Anand, P.N. Leigh, N. Cairns, Neurotrophin-3-like immunoreactivity in human spinal motoneurones in Amyotrophic Lateral Sclerosis, J. Neurol. Sci. 148 Ž1997. 33–40. w8x A. Eisen, S. Kim, B. Pant, Amyotrophic lateral sclerosis ŽALS.: a phylogenetic disease of the corticomotoneuron?, Muscle Nerve 15 Ž1992. 219–224. w9x J. Ekblom, T. Ebendal, I. Gillberg, S. Aquilonius, Quantitative autoradiography of high-affinity nerve growth factor receptors in various segments of the human spinal cord fails to demonstrate alterations in amyotrophic lateral sclerosis, Neurodegeneration 1 Ž1992. 163–168. w10x A. Ghosh, J. Carnahan, M.E. Greenberg, Requirement for BDNF in activity-dependent survival of cortical neurons, Science 263 Ž1994. 1618–1623. w11x C.E. Henderson, W. Camu, C. Mettling, A. Gouin, K. Poulsen, M. Karihaloo, J. Rullamas, T. Evans, S.B. McMahon, M.P. Armanini et al., Neurotrophins promote motor neuron survival and are present in embryonic limb bud, Nature 363 Ž1993. 266–270. w12x C.E. Henderson, H.S. Phillips, R.A. Pollock, A.M. Davies, C. Lemeulle, M. Armanini, L. Simmons, B. Moffet, R.A. Vandlen, L.C. Simpson, GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle, Science 266 Ž1994. 1062– 1064. w13x Z. Kokaia, J. Bengzon, M. Metsis, M. Kokaia, H. Persson, O. Lindvall, Coexpression of neurotrophins and their receptors in neurons of the central nervous system, Proc. Natl. Acad. Sci. USA 90 Ž1993. 6711–6715. w14x P.A. Lapchak, D.M. Araujo, K.D. Beck, C.E. Finch, S.A. Johnson, F. Hefti, BDNF and trkB mRNA expression in the hippocampal formation of aging rats, Neurobiol. Aging 14 Ž1993. 121–126. w15x P.N. Leigh, K. Ray-Chadhuri, Motor neuron diseases, J. Neurol. Neurosurg. Psychiatry 57 Ž1994. 886–896.
R.M. Duberley et al.r Brain Research 763 (1997) 259–263 w16x R.M. Lindsay, Neurotrophins and receptors, Prog. Brain Res. 103 Ž1994. 3–14. w17x R. Pamphlett, J. Kril, T.M. Hng, Motor neuron disease: a primary disorder of corticomotoneurons?, Muscle Nerve 18 Ž1995. 314–318. w18x E. Pioro, J.P. Antel, N.R. Cashman, D.L. Arnold, Detection of cortical neuron loss in motor neuron disease by proton magnetic resonance spectroscopic imaging in vivo, Neurology 44 Ž1994. 1933–1938. w19x R. Porter, R. Lemon, Corticospinal function and voluntary movement, Monographs of The Physiological Society 45, Clarendon Press, Oxford, 1993. w20x J.S. Rudge, Astrocyte-derived neurotrophic factors, in: S. Murphy ŽEd.. Astrocytes, Pharmacology and Function, Academic Press, London, 1993, pp. 267–305. w21x L. Schnell, Neurotrophin-3 enhances sprouting of corticospinal tract during development and after adult spinal cord lesion, Nature 367 Ž1994. 170–173. w22x M. Schorr, L. Zhou, K. Schwechheimer, Expression of ciliary neurotrophic factor is maintained in spinal motor neurons of amyotrophic lateral sclerosis, J. Neurol. Sci. 140 Ž1996. 117–122. w23x J.L. Seeburger, S. Tarras, H. Natter, J.E. Springer, Spinal cord motoneurons express p75 NG FR and trkB mRNA in amyotrophic lateral sclerosis, Brain Res. 621 Ž1993. 111–115. w24x M. Sendtner, B. Holtmann, R. Kolbeck, H. Thoenen, Y.A. Barde, Brain-derived neurotrophic factor prevents the death of motoneurons in newborn rats after nerve section, Nature 360 Ž1992. 757–759. w25x M. Sendtner, G.W. Kreutzberg, H. Thoenen, Ciliary neurotrophic factor prevents the degeneration of motor neurons after axotomy, Nature 345 Ž1990. 440–441.
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w26x N.A. Seniuk-Tatton, J.T. Henderson, J.C. Roder, Neurons express ciliary neurotrophic factor mRNA in the early postnatal and adult rat brain, J. Neurosci. Res. 41 Ž1995. 663–676. w27x G.T. Serratrice, T. Munsat, Pathogenesis and Therapy of Amyotrophic Lateral Sclerosis, Lippincott–Raven, Philadelphia, 1995. w28x S. Shu, G. Ju, L. Fan, The glucose-DAB-nickel method in peroxidase histochemistry of the nervous system, Neurosci. Lett. 85 Ž1988. 169–171. w29x J.E. Springer, X. Mu, L.W. Bergmann, J.Q. Trojanowski, Expression of GDNF mRNA in rat and human nervous tissue, Exp. Neurol. 127 Ž1994. 167–170. w30x H. Thoenen, Neurotrophins and neuronal plasticity, Science 270 Ž1995. 593–598. w31x A. Tomac, J. Widenfalk, L.F. Lin, T. Kohno, T. Ebendal, B.J. Hoffer, L. Olson, Retrograde axonal transport of glial cell line-derived neurotrophic factor in the adult nigrostriatal system suggests a trophic role in the adult, Proc. Natl. Acad. Sci. USA 92 Ž1995. 8274–8278. w32x C.S. von Bartheld, M.R. Byers, R. Williams, M. Bothwell, Anterograde transport of neurotrophins and axodendritic transfer in the developing visual system, Nature 379 Ž1996. 830–833. w33x C.S. von Bartheld, R. Williams, F. Lefcort, D.O. Clary, L.F. Reichardt, M. Bothwell, Retrograde transport of neurotrophins from the eye to the brain in chick embryos: roles of the p75NTR and trkB receptors, J. Neurosci. 16 Ž1996. 2995–3008. w34x Q. Yan, R.D. Rosenfeld, C.R. Matheson, N. Hawkins, O.T. Lopez, L. Bennet, Expression of Brain-derived neurotrophic factor ŽBDNF. protein in the adult rat central nervous system, Neuroscience Ž1997. Žin press..
Short communication
Immunocytochemical studies of neurotrophins in cerebral motor cortex in Amyotrophic Lateral Sclerosis R.M. Duberley a
a,b
, I.P. Johnson
a, )
, P. Anand b , P.N. Leigh c , N.J. Cairns
d
Department of Anatomy and DeÕelopmental Biology, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, UK Department of Neurology, Saint Bartholomew’s and The Royal London Hospital School of Medicine and Dentistry, London E1 lBB, UK c Department of Clinical Neuroscience, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK d Department of Neuropathology, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK
b
Accepted 1 April 1997
Abstract Neurotrophin-like immunoreactivity was studied in post-mortem motor cerebral cortex from patients with Amyotrophic Lateral Sclerosis ŽALS. and controls. Neurotrophin-4r5 immunoreactivity was seen in small-Ž12–25 mm., medium-Ž26–39 mm., and large-Ž) 40 mm., neurones, neurotrophin-3 was seen in medium and small neurones, while brain-derived neurotrophic factor was restricted to small neurones. No difference in number or intensity of immunostained neurones was found between ALS and controls. q 1997 Elsevier Science B.V. Keywords: Amyotrophic lateral sclerosis; Motor cortex; Neurotrophic factor; Immunocytochemistry
Amyotrophic lateral sclerosis ŽALS. is a neurodegenerative disorder characterised by progressive muscle weakness, combined with symptoms of corticospinal tract degeneration w27x. Motoneurones in the ventral horn of the spinal cord are lost, as are brainstem motoneurones and pyramidal neurones of the cerebral motor cortex; the number of motoneurones lost in these regions varies with the clinical sub-type of the disease w15,18x. The aetiology of the most common form of this disease Žsporadic ALS. remains unknown. Nevertheless, hopes for a therapy have been raised by recent animal studies which have demonstrated the survival-promoting properties of neurotrophic factors on spinal motoneurones w3,11,12,24,25x, prompting clinical trials of systemically-administered neurotrophic factors, such as brain-derived neurotrophic factor ŽBDNF. and insulin-like growth factor-1 ŽIGF-1. and ciliary neurotrophic factor ŽCNTF.. We found previously that levels of CNTF, but not nerve growth factor ŽNGF., were decreased in the ventral horn of ALS spinal cord w2,6x. More recently, we found that ALS spinal motoneurones had reduced neurotrophin-3 ŽNT-3.-like immunoreactivity compared to controls w7x. Taken together with reports of
)
Corresponding author. Fax q44 Ž171. 830-2917.
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 4 6 5 - 4
changes in the receptors for IGF- 1, BDNF and CNTF in spinal motoneurones in ALS w1,6,23x, these observations serve to emphasise the potential importance of neurotrophic factors in ALS. All these studies are, however, concerned with the spinal cord and lower motoneurones, and the extent to which neurotrophic factors or their receptors are involved in upper motoneuronal loss remains unclear. We have previously found that mRNA levels of both CNTF the a subunit of the CNTF receptor in the motor cerebral cortex are unchanged in ALS w2,6x, with very low levels of CNTF measured by ELISA in motor and occipital cerebral cortex in comparison with the spinal cord. Interestingly, NGF levels are decreased in ALS motor cortex, but not occipital cortex w2x, and increased levels are found in the dorsolateral fasciculus of the spinal cord where degenerating corticospinal fibres are found. Little is known, however, of the expression of other neurotrophins in ALS. In this study, therefore, we have used immunocytochemical methods to study the neurotrophins NT-3, neurotrophin 4r5 ŽNT-4r5. and BDNF in postmortem human motor cortices of control and ALS patients. Motor cerebral cortex was obtained post-mortem from eight patients aged 58–71 years, clinically diagnosed with sporadic ALS, and 5 age-matched control patients Ž63–83 years. with no clinical history of neurological disease. The
260
R.M. Duberley et al.r Brain Research 763 (1997) 259–263
mean post-mortem interval for ALS patients was 18.8 h Žrange 4–24 h. and for control patients was 23.4 h Žrange 19–26 h.. Tissue was snap-frozen on a brass plate at y708C and stored at y708C until required. The presence of large Ž) 40 mm. pyramidal neurones in cortical layer V of cryostat sections stained with 0.05% Cresyl fast violet was taken as confirmation that the sections were from the motor cortex region. As expected, fewer Betz cells were found in ALS tissue. After confirming the identity of the
tissue block, cryostat sections Ž20 mm. were mounted on chrome-alum-subbed slides, allowed to equilibrate to room temperature, then fixed for 30 min in 4% paraformaldehyde in phosphate buffered saline ŽPBS., pH 7.4. After rinsing twice in 0.1 M PBS, pH 7.4, slides were incubated in 0.2% Triton X-100 in PBS for 1 h to improve the access of antibodies to tissue antigens. Slides were then washed in PBS and incubated with 0.05% H 2 0 2 in PBS for 20 min to block endogenous peroxidase activity. Slides were again
Fig. 1. NT-3, NT-4 and BDNF-like immunoreactivity in the motor cortex of control and ALS patients. Scale bar s 40 mm.
R.M. Duberley et al.r Brain Research 763 (1997) 259–263
washed in PBS prior to a 20 min incubation with normal goat or horse serum diluted 1:30 in PBS to reduce background immunostaining. Sections were blotted to remove excess blocking serum and then incubated with the primary antibody in PBS containing 0.03% Triton X-100, 0.1% sodium azide and 0.1% bovine serum albumin ŽBSA. for 24 h at 48C. The BDNF antibody was raised in rabbits against E. coli-derived recombinant human BDNF and shown not to cross-react with nerve growth factor, NT-3 or NT-4 w34x. Similar, but unpublished characterisation has been carried out for the NT-3 Žpers. commun., J. Carnaham, Amgen. and NT-4r5 antibodies. The NT-4r5 staining was also confirmed using a another, commerciallyavailable, polyclonal antibody ŽSanta Cruz Biotechnology, Inc., USA, dilution: 1:1K. raised to a 20 amino acid peptide sequence of the mature form of human NT-4r5 Ždata not shown.. The working dilutions, source and reference numbers of the primary antibodies used in this study are summarised below: Polyclonal BDNF Ž1:5K. ŽAmgen Inc. USA., ŽRef. 7253-34 ŽA..; Polyclonal NT-3 Ž1:lOK. ŽAmgen Inc. USA., ŽRef. 8Cr845 a883.; Polyclonal NT-4r5 Ž1:lK. ŽDr. D. Kaplan, Advanced BioScience Labs.., ŽRef. a NT-4. After incubation with the primary antibody, sections were rinsed in 0.025% Tween 20rPBS, washed in PBS and then incubated in the biotinylated secondary antibody at a dilution of 1:100 in PBS for 1 h. Following a further rinse in 0.025% Tween 20rPBS and three PBS washes, the sections were incubated for 1 h in an avidin-biotin ŽABC. complex Ž1: 100 in PBS containing 0.1% BSA without azide; Vector Labs... Slides were again rinsed in 0.025% Tween 20rPBS, followed by three washes in PBS. Staining was then visualised by incubating the slides with 0.05% diaminobenzidine containing 2.5% nickel sulphate and 0.001% glucose oxidase w28x for 5 to 15 min. Sections were then dehydrated and mounted in DPX. Immunostaining was abolished by overnight preabsorption of the antibody at 48C with 100 ng-200 mgrml of the corresponding antigen Žrecombinant human BDNF, NT-3 or NT-4r5, Amgen.. Omission of primary or secondary antibodies produced no staining, while preabsorption of the
261
antibody with inappropriate antigens had no effect on immunostaining. Using a =40 objective lens and a X10 eyepiece, the numbers and mean diameters Žmax q minr2. of immunostained and non-immunostained neurones in randomlyplaced fields of 855 mm2 area, over layer V of the motor cortex were determined without knowledge of the slide codes for 4–8 slides per subject. For each neurotrophin, the percentage of all neurones showing neurotrophin-like immunoreactivity was determined. NT-3-like immunoreactivity was detected in small Ž12– 25 mm. and medium Ž26–39 mm. diameter neurones within the motor cortex ŽFig. 1., but not in large neurones. No obvious differences of staining intensity or numbers of immunopositive cells in control Ž49.0 " 10.0% positive cells. and ALS patients Ž52.5 " 5.4% positive cells. were seen ŽTable 1.. NT-4r5-like immunoreactivity using both sources of primary antibody was present within small and medium-diameter neurones Žrange: 12–32 mm. in the motor cortex of control Ž51.0 " 10.2% positive cells. and ALS patients Ž49.1 " 11.9% positive cells.. Occasional staining of larger pyramidal neurones Ž49–55 mm diameter. of both control and ALS patients was also seen. Some small Ž- 10 mm. presumptive neuroglial cells within the motor cortex were also immunopositive for both NT-3 and NT-4r5. Occasional weak BDNF-like immunostaining of small neurones Ždiameter range: 12–28 mm. within the motor cortices of control Ž21.3 " 7.7% positive cells. and ALS patients Ž21.7 " 9.9% positive cells. was seen. Our immunocytochemical studies have shown that, in general, neurotrophic factors are localised to the cell bodies of small and medium-sized neurones of the cerebral motor cortex, and to presumptive neuroglia. Both neurones and neuroglia synthesise a variety of neurotrophic factors w13,14,20,26,29,30x and these can be taken up and transported retrogradely or anterogradely by neurons w16,31– 33x. In the absence of additional in situ hybridisation studies to define the cellular location of the mRNAs, however, we cannot estimate the extent to which our immunocytochemical findings reflect the synthesis of neurotrophic factors by the labelled cells or uptake from other sources. Such information could also help rationalise some apparent discrepancies between reported levels of neu-
Table 1 Mean number of neurones and the proportion showing neurotrophin-like immunoreactivity within area V of the motor cerebral cortex of five control and eight ALS patients Control
BDNF NT-3 NT-4
Mean S.D. Mean S.D. Mean S.D.
ALS
Total
Positive
% Positive
Total
Positive
% Positive
16.4 5.8 14.9 4.6 10.0 1.3
3.5 0.6 7.3 2.3 5.1 1.1
21.3 7.7 49.0 10.0 51.0 10.2
14.3 4.8 20.2 6.1 16.7 5.6
3.1 1.0 10.6 4.0 8.2 3.1
21.7 9.9 52.5 5.4 49.1 11.9
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rotrophic factors and their receptors. Thus, increased expression of trkB and p75 NG FR has been found for motoneurones in ALS spinal cord w23x, without increased NGF binding w9x or increased immunostaining of motoneurones for NGF or BDNF Žunpubl. obs... Large-diameter pyramidal neurones of the motor cortex have classically been equated with Betz cells, which contribute axons to the corticospinal tract. In contrast to large-diameter spinal motoneurones w6,22x, however, we found little staining of presumptive Betz cells for neurotrophic factors, and no obvious changes in ALS. This finding may be a reflection of the low number of Betz cells seen normally in single sections in the present study and the further sampling problems imposed by their loss in ALS. Alternatively, the absence of neurotrophic factor changes in Betz cells could be taken to indicate that mechanisms other than those involving neurotrophic factors contribute towards their loss. This proposal does not preclude the involvement of neurotrophic factors in the loss of medium and small neurones. It is likely that many of these smaller neurones contributed to the corticospinal tract, given that the Betz cell contribution may account for less that 5% of fibres w19x. Other immunolabelled cortical neurones observed in this study may have been local interneurones. Their persistence in the face of a reduction in the reference area due to the general atrophy of layer V may explain our finding that the density of neurones of all sizes was slightly increased in ALS cortex compared to controls ŽTable 1.. Animal studies have demonstrated the importance of neurotrophic factors in promoting the survival and sprouting of injured corticospinal neurones w5,21x, as well as the structural and functional development of the visual cortex w10,30x. As both situations involve modifications of the connectivity of cortical interneurones and projection neurones, it is possible that one role of neurotrophic factors in the cerebral motor cortex is to maintain synaptic connectivity. Such a role assumes the intraneuronal transport of neurotrophic factors, and it is interesting that defective axonal transport associated with overexpression of the human heavy neurofilament subunit gene has recently been shown in a transgenic mouse model of ALS w4x. In the context of theories on the pathogenesis of ALS w8,17x, it will be important, if suitable markers of interneurones and upper motoneurones can be found, to determine the relative proportion of corticospinal neurones to interneurones which are immunopositive for neurotrophic factors and the relative degree of neuronal degeneration occurring in these two populations.
Acknowledgements This work was supported by a grant from the Motor Neurone Disease Association, UK. The NT-4r5 antibody was a gift from Dr. D. Kaplan ŽAdvanced BioScience
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