Brain-derived neurotrophic factor is reduced in Alzheimer's disease

Molecular Brain Research 49 Ž1997. 71–81

Brain-derived neurotrophic factor is reduced in Alzheimer’s disease B. Connor a , D. Young a , Q. Yan c , R.L.M. Faull b, B. Synek d , M. Dragunow a

a,)

Department of Pharmacology, Faculty of Medicine and Health Science, UniÕersity of Auckland, Auckland New Zealand b Department of Anatomy, Faculty of Medicine and Health Science, UniÕersity of Auckland, Auckland, New Zealand c Amgen, Thousand Oaks, CA, USA d Department of Pathology, Faculty of Medicine and Health Science, UniÕersity of Auckland, Auckland, New Zealand Accepted 18 March 1997

Abstract Alzheimer’s disease may be due to a deficiency in neurotrophin protein or receptor expression. Consistent with this hypothesis, a reduction in BDNF mRNA expression has been observed in human post-mortem Alzheimer’s disease hippocampi. To further investigate this observation, we examined whether the alteration in BDNF expression also occurred at the protein level in human post-mortem Alzheimer’s disease hippocampi and temporal cortices using immunohistochemical techniques. We observed a reduction in the intensity and number of BDNF-immunoreactive cell bodies within both the Alzheimer’s disease hippocampus and temporal cortex when compared to normal tissue. These results support and extend previous findings that BDNF mRNA is reduced in the human Alzheimer’s disease hippocampus and temporal cortex, and suggest that a loss of BDNF may contribute to the progressive atrophy of neurons in Alzheimer’s disease. q 1997 Elsevier Science B.V. Keywords: Brain-derived neurotrophic factor; Alzheimer’s disease; Hippocampus; Neurodegeneration; Down’s syndrome

1. Introduction Brain-derived neurotrophic factor ŽBDNF. is a member of the structurally and functionally homologous neurotrophic family which also consists of nerve growth factor ŽNGF., neurotrophin-3 ŽNT-3. and neurotrophic factor-4r5 ŽNT-4r5.. BDNF mRNA and protein levels have been detected in the hippocampus, amygdala, projection areas of the olfactory system, inner and outer pyramidal layers of the neocortex, claustrum, cerebellum and the superior colliculus w21,23,28,35,56,64,70,75,76 x suggesting that BDNF has a more widespread distribution than NGF. Within both the rat and human hippocampus, BDNF mRNA and protein levels have been visualized in the hilar region of the dentate gyrus and the pyramidal and apical dendritic processes of the CA3, CA2, CA1 and subiculum regions w21,64,79x. Members of the neurotrophin family have been proposed to play a role in the protection of specific neuronal

) Corresponding author. Department of Pharmacology, Faculty of Medicine and Health Science, University of Auckland, Private Bag 92019, Auckland, New Zealand. Fax: q64 Ž9. 373-7556.

0169-328Xr97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 1 2 5 - 3

populations by suppressing the expression of ‘suicide genes’ which, when activated are involved in the process of programmed cell death w2,32,60x. BDNF has been observed to either stimulate the differentiation or enhance the survival of cholinergic neurons both in vitro w37,48x and in vivo w41x. In the adult rat brain, hippocampal damage has been reported to up-regulate BDNF mRNA expression within injury-resistant regions of the hippocampus w6,12,13,27,38,45,62,71,73 x. Furthermore, BDNF has been reported to increase the survival of basal forebrain cholinergic neurons after fimbrial transection w54x and excitotoxic lesioning w11x. The administration of rhBDNF to rats after fimbrial transection has been observed to result in a reduction in axotomy-induced degeneration of the basal forebrain cholinergic neurons w37x and to induce the sprouting of cholinergic processes within the residual hippocampal neurons ipsilateral to the lesion w11x. The observed trophic effects of BDNF on several different neuronal populations after brain insults has lead to the suggestion that BDNF may have a protective effect on neuronal systems in neurodegenerative disorders such as Alzheimer’s disease. Alzheimer’s disease is a degenerative disorder of the central nervous system characterized by senile plaques, neurofibrillary tangles and b-amyloid deposition. Selective

B. Connor et al.r Molecular Brain Research 49 (1997) 71–81

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neuronal loss occurs in Alzheimer’s disease in the areas of the nucleus basalis of Meynert, locus coeruleus, hippocampus, amygdala, and neocortical association areas w17,50x. It has been proposed that Alzheimer’s disease pathology may be due to a deficit in neurotrophin protein or receptor expression w3,25x. While there is little evidence supporting this proposal, a reduction in BDNF mRNA expression has been observed in human post-mortem Alzheimer’s disease hippocampi when compared to normal hippocampal levels w54,63x. While the level of BDNF mRNA expression in the human post-mortem Alzheimer’s disease hippocampus has been examined, it has not been investigated whether this observed alteration in BDNF expression also occurs at the protein level. Using a polyclonal antibody directed against the BDNF polypeptide, we compared the level of BDNF protein in human post-mortem Alzheimer’s disease and neurologically normal hippocampal and temporal cortex sections using immunohistochemistry techniques.

2. Materials and methods 2.1. Human post-mortem brain tissue All studies using human tissue were performed under approval from the University of Auckland Human Ethical Committee. The normal and Alzheimer’s disease brain tissue used in this study were obtained from the New Zealand Neurological Foundation Human Brain Bank in

the Department of Anatomy, University of Auckland. Alzheimer’s disease brain tissue under went pathological examination to confirm clinical diagnosis based on the CERAD neuropathological protocol w52x. Alzheimer’s disease cases were matched with normal cases of a similar age and post-mortem delay ŽTable 1.. 2.2. Anti-BDNF antibody 10 New Zealand white rabbits were hyperimmunized over a period of 4 months with E. coli-derived recombinant human BDNF, covalently coupled to keyhole limpet hemocyanin. After 4 months of boosting and monitoring, bleeds revealed high anti-BDNF antibody titers in all 10 rabbits antisera. Production bleeds of f 50 ml were obtained from each animal and pooled together to serve as the source for subsequent affinity purification procedures. The antibody was affinity-purified over a BDNF column. Cross-reactivity with other neurotrophins was removed by sequentially passing the antibody over NGF, NT-3 and NT-4r5 columns. Characterization of the antibody using Western blotting showed that it was specific to BDNF but not the other members of the neurotrophic family. The neutralizing activity of the antibody to BDNF but not the other neurotrophins was demonstrated by dorsal root ganglion explant assays for neurite outgrowth and dissociated dorsal root ganglion neurons for cell survival. In addition, this antibody did not stain BDNF knock-out mouse brains and showed a staining pattern in wild-type mice brains similar to that seen in rat brains w79x.

Table 1 Post-mortem data of neurologically normal and Alzheimer’s disease brain tissue used in BDNF immunohistochemistry study Case number

Age Žyears.

Sex

Cause of death

Post-mortem delay Žh.

H69 a H72 H68 H98 H71 H74 H73 H70 AZ13 d AZ14 AZ15 AZ17 AZ19 AZ20 AZ22 AZ23 AZ25 DO1 f

47 52 55 60 82 65 73 83 68 77 80 61 73 72 81 71 73 43

M M M M M F M M F F M F F F F F M M

IHD b IHD IHD MI c IHD MI IHD IHD Heart failure during surgery Bronchopneumonia Pneumonia Pneumonia AD e AD Bronchopneumonia Bronchopneumonia Bronchopneumonia MI

22 10.75 14 18 9 5 9 9 16 15 3.75 34 16 3 2 10 47 18

a b c d e f

H, neurologically normal human brain section. IHD, ischemic heart disease. MI, myocardial infarction. AZ, Alzheimer’s disease human sections. AD, Alzheimer’s disease. DO, Down’s syndrome.

B. Connor et al.r Molecular Brain Research 49 (1997) 71–81

2.3. Immunohistochemistry Formalin-fixed temporal lobes and hippocampi from neurologically normal post-mortem controls Ž1 female and 7 males, mean age s 64.6 years, mean post-mortem delay s 12 h., Alzheimer’s disease Ž7 females and 2 males, mean age s 72.8 years, mean post-mortem delay s 16.5 h. and Down’s syndrome Ž1 male, age s 43 years, postmortem delay s 18 h. brains were cryoprotected in 30% sucrose solution in 0.1 M phosphate buffer and cut on a sledge microtome at 50 mm. The sections were then treated with 1% hydrogen peroxide in 50% methanol for 10 min and washed in PBS for a further 10 min. The polyclonal antibody to BDNF Ždiluted 1 : 20 000 with immunobuffer. was applied to the sections and incubated with gentle agitation for 72 h at 48C. The primary antibody was removed and washed in PBS for 10 min before being incubated overnight in a biotinylated anti-rabbit secondary antibody. Immunostaining was visualized using an extravidinrhorseradish peroxidase-diaminobenzidine ŽDAB . method as previously described w20x. 2.4. Western blotting – ECL technique Neurologically normal human hippocampal and temporal lobe blocks were dissected out and stored at y808C until required. Tissue homogenates were prepared by homogenization of thawed tissue in 500 m l of 0.25 M sucrose containing 10 mm Tris-HCl pH 7.4, 1 mm EDTA and 17 mgrml PMSF using a Ultraturrax homogenizer ŽJanke and Kunckel. and centrifuged at 10 000 = g for 15 min at 48C. The supernatant was removed and protein concentrations were determined using the Biorad protein assay as described by the manufacturers. The protein samples Ž40 m g concentration. were separated by SDS-polyacrylamide electrophoresis using a 15% Žwrv. acrylamide resolving gel under reducing conditions. The separated protein was electrophoretically transferred onto a nitrocellulose membrane. The membrane was blocked by incubation with 1% Žwrv. bovine serum albumin ŽBSA. in Tris-buffered saline containing 10% Žwrv. normal goat serum ŽNGS. for 30–40 min at room temperature with gentle agitation. The sample lane was incubated for 24 h at 48C with the polyclonal antibody to BDNF Ždiluted 1 : 1000 in TBST containing 1% BSA and 10% NGS. as described in the immunohistochemistry section above. After washing in TBST Ž3 = 5 min., the standard lane was incubated in extravidin peroxidase Ždiluted 1 : 500 in TBST. while the sample lane was incubated in a peroxidase-linked, secondary biotinylated donkey anti-rabbit antibody ŽAmersham, 1 : 1500 in TBST. at 48C for 3 h with gentle agitation. In order to control for non-specific staining, an additional lane containing normal hippocampal protein ŽH77. was incubated only in the peroxidase-linked, secondary donkey anti-rabbit antibody Ž1 : 1500 in TBST. at 48C for 12 h with gentle agitation. The immunoblot was

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then washed in TBST Ž3 = 5 min. and the protein bands visualized using the ECL method ŽAmersham.. To verify the results seen with the BDNF primary antibody ŽAmgen., normal ŽH77. hippocampal and temporal lobe samples were separated by SDS-polyacrylamide electrophoresis and incubated for 24 h at 48C with a different polyclonal antibody to BDNF ŽN-20, Santa Cruz Biotechnology, Cat. No. sc-546, diluted 1 : 1000 in TBST containing 1% BSA and 10% NGS.. The immunoblot was visualized as described above. 2.5. Quantification BDNF immunoreactivity was analysed using the MD30 Image Analysis system ŽLeading Edge, Australia. and video camera mounted on a Leitz Diaplan microscope. Cell counts were taken from randomly selected regions of the Alzheimer’s disease and normal CA1 hippocampal region and temporal cortex and the overall means determined. By capturing the entire hilar region, the mean density of BDNF immunoreactivity was determined for both the Alzheimer’s disease and normal brains. Statistical analysis was performed on each of these regions using an unpaired Student’s t-test.

3. Results 3.1. BDNF immunohistochemistry The level of BDNF immunoreactivity was examined in regions of the neurologically normal Ž n s 8. and Alzheimer’s disease Ž n s 9. hippocampus and temporal cortex using a polyclonal antibody directed against BDNF. In normal human hippocampal sections, a high level of diffuse BDNF immunostaining was observed in the hilar region of the dentate gyrus. In addition BDNF-immunopositive cell bodies were detected within the granule cell layer of the dentate gryus. In the CA3 and CA1 regions of the normal hippocampus, strong BDNF immunoreactivity was seen in the pyramidal cell bodies and dendritic processes ŽFig. 1A,C.. BDNF-immunopositive cell bodies were also observed within layers II–VI of the normal temporal cortex ŽFig. 1E.. In contrast, the level of BDNF immunoreactivity observed in Alzheimer’s disease hippocampal sections was greatly reduced when compared to the normal brain. Both the number of BDNF-immunopositive neurons and the intensity of immunostaining within cell bodies was reduced within regions of the Alzheimer’s disease brain. Little or no cell body immunoreactivity was observed within either the granule cell layer of the dentate gryus or the pyramidal cell layer of the CA1 to CA3 subregions in the Alzheimer’s disease hippocampus ŽFig. 1B,D.. A reduction in the level of BDNF immunoreactivity was also seen in the hilar region of the dentate gryus in Alzheimer’s

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B. Connor et al.r Molecular Brain Research 49 (1997) 71–81

disease hippocampal sections ŽFig. 2.. In addition, a reduction in both the number and intensity of BDNF-immunopositive neurons was observed in the Alzheimer’s disease temporal cortex when compared to the normal human cortex ŽFig. 1F.. The mean number of BDNF-immunoreactive cell bodies were determined in randomly selected sections of the CA1 hippocampal region and the temporal cortex in both the normal and Alzheimer’s disease brain. The results

revealed that the mean number of BDNF-immunopositive cell bodies in both the CA1 hippocampal region and temporal cortex of the Alzheimer’s disease brain was greatly reduced when compared to normal sections ŽFig. 4.. While analysis revealed that the difference in the mean number of BDNF-positive cell bodies in the CA1 region of the Alzheimer’s disease hippocampus was not significantly different to that seen in the normal CA1 region Ž P F 0.2609., the level of BDNF immunoreactivity observed in

Fig. 1. The level of BDNF immunostaining in the neurologically normal ŽA,C,E. and Alzheimer’s disease ŽB,D,F. post-mortem human hippocampus ŽA–D. and temporal cortex ŽE,F.. ŽA. A high level of BDNF immunoreactivity was observed in the CA1 pyramidal neurons in the normal human hippocampus while in the CA1 region of the Alzheimer’s disease hippocampus; ŽB. the level of immunoreactivity was greatly reduced with only a few cell bodies staining positive for BDNF Žfilled arrowhead.; ŽC. in the CA3 region of the normal human hippocampus, a strong level of BDNF immunoreactivity was observed within the cell bodies of the pyramidal neurons Žfilled arrowhead.. ŽD. On comparison, the level of BDNF immunoreactivity detected in the pyramidal cell bodies in the Alzheimer’s disease CA3 hippocampal region was greatly reduced Žopen arrowhead.; ŽE. BDNF immunopositive cell bodies were observed within layers II to VI of the neurologically normal human temporal cortex. In the Alzheimer’s disease temporal cortex ŽF. a reduction in the intensity and mean number of BDNF-immunopositive cell bodies was detected in all cortical layers Žfilled arrowhead.. Scale bar s 100 m m.

B. Connor et al.r Molecular Brain Research 49 (1997) 71–81

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Fig. 2. The level of BDNF immunostaining in the hilar region of the neurologically normal ŽA. and Alzheimer’s disease ŽB. post-mortem human hippocampus. A high level of BDNF immunoreactivity was observed in the hilar regions of the neurologically normal brain ŽA.. In comparison, a reduction in the density of BDNF immunostaining was seen in the hilar region of the dentate gyrus in Alzheimer’s disease hippocampal sections. Scale bar s 200 m m.

the Alzheimer’s disease temporal cortex was significantly less than that seen in normal sections Ž P F 0.0456.. The lack of significant alteration in the level of BDNF immunoreactivity reported in the CA1 region of the Alzheimer’s disease hippocampus may reflect the large standard error seen in the normal sample. This may be due to the variation in BDNF immunoreactivity observed among the individual normal cases examined and may also suggest the presence of pathological processes at a subclinical level. In addition, the mean density of BDNF immunoreactivity was determined in the hilar region of the normal and Alzheimer’s disease hippocampus. Analysis indicated that the mean density of BDNF immunoreactivity in the hilar region of the Alzheimer’s disease hippocampus was significantly reduced Ž P " 0.04. when compared to the normal hilar region ŽFig. 3.. In addition to the Alzheimer’s disease hippocampal sections, we also investigated the level of BDNF immunoreactivity in the hippocampus of a Down’s syndrome case

Fig. 3. Graph comparing the mean density Ž"S.E.M.. of BDNF immunoreactivity within the hilar region of the dentate gyrus in the Alzheimer’s disease Ž ns9. and neurologically normal human Ž ns8. hippocampus. ) The difference in the mean density is considered significant using a two-tailed t-test with 15 df. The P value is F 0.04.

ŽDO1.. Down’s syndrome patients over the age of 30 years have been reported to develop b-amyloid plaques and neurofibrillary tangles similar to those observed in Alzheimer’s disease w9,10,24,42,51,55,58,66x and the maturation of these pathological features appear to be identical

Fig. 4. Graph comparing the mean cell count Ž"S.E.M.. of BDNF-immunopositive cell bodies within the CA1 subregion ŽA. and the temporal cortex ŽB. of the Alzheimer’s disease Ž ns9. and neurologically normal human Ž ns8. brain. ŽB. ) The difference in the mean cell count is considered significant using a two-tailed t-test with 14 df. The P value is F 0.0456.

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B. Connor et al.r Molecular Brain Research 49 (1997) 71–81

to that observed in Alzheimer’s disease w8x. Supporting this similarity in pathological changes between Alzheimer’s disease and Down’s syndrome patients, we observed that, like the Alzheimer’s disease hippocampal sections, the intensity and number of BDNF-immunoreactive cell bodies in regions of the Down’s syndrome hippocampus and temporal cortex were greatly reduced when compared to normal brain tissue. 3.2. Post-mortem delay In order to determine whether the mean post-mortem delay observed for the Alzheimer’s disease cases examined in this study Žmean post-mortem delay s 16.5 " 4.5 h. was significantly greater than that reported for the normal cases Žmean post-mortem delay s 12 " 1.9 h., an unpaired Student’s t-test was performed. Analysis indicated that there was no significant difference between the mean postmortem delay observed for each group Ž P F 0.43.. In addition, a Pearson’s linear correlation test was performed in order to determine whether the level of BDNF immunoreactivity observed in the Alzheimer’s disease hippocampus and temporal cortex was effected by post-mortem delay. The mean number of BDNF-immunopositive neurons within the CA1 hippocampal region and the temporal cortex of the Alzheimer’s brain were compared with the post-mortem delay for each case. The correlation between post-mortem delay and the mean density of BDNF immunostaining in the hilar region of the Alzheimer’s disease hippocampus was also examined. No correlation was observed between the mean number of BDNF-immunopositive neurons and post-mortem delay in either the CA1 hippocampal region Ž P F 0.6884, r s

Fig. 6. Western blot analysis of the BDNF protein using the Santa Cruz polyclonal antibody in post-mortem normal human brain ŽH77. protein samples. A BDNF-immunoreactive band was detected at 14 kDa in both the normal temporal cortex Žtc. and hippocampus Žhp.. This band was not seen in the secondary antibody control lane Žc.. Molecular weight marker in kDa is indicated on the right. A standard lane ŽM. is indicated on the left.

0.02436. or the temporal cortex Ž P F 0.6884, r s 0.02436. in the Alzheimer’s disease brain. Furthermore, no correlation was observed between the density of BDNF immunostaining in the hilar region of the Alzheimer’s disease hippocampus and post-mortem delay Ž P F 0.2438, r s 0.1879.. These results indicate there is no direct relationship between the reduction of BDNF immunoreactivity in the Alzheimer’s disease brain and post-mortem delay. Therefore, post-mortem delay cannot account for the observed reduction in BDNF immunoreactivity detected in the Alzheimer’s disease brain. 3.3. Western blotting

Fig. 5. Western blot analysis of the BDNF protein ŽAmgen. in human post-mortem neurologically normal protein samples. Two BDNF-immunopositive bands were detected in the normal ŽH77. temporal cortex Žtc. and hippocampal Žhp. protein samples at 14 and 28 kDa. Neither of the immunopositive bands were detected in the secondary antibody control lane, although a number of non-specific high molecular weight bands were detected Žc.. Molecular weight markers in kDa are indicated on the right. A standard lane ŽM. is indicated on the left.

Using protein samples derived from the neurologically normal ŽH77. human hippocampus and temporal cortex, Western blotting was performed in order to confirm the specificity of the primary BDNF antibody within the human post-mortem brain. In the normal ŽH77. human hippocampus and temporal cortex protein samples, the BDNF antibody detected two immunopositive bands – a strong band at f 28 kDa and a fainter band at f 14 kDa ŽFig. 5.. In order to determine the specificity of these immunopositive bands, a secondary control study was performed in which the normal human hippocampal sample ŽH77. was incubated with the biotinylated secondary donkey anti-rabbit antibody. Incubation of the normal hippocampal sample in the secondary donkey anti-rabbit antibody did not detect either the 28- or the 14-kDa bands ŽFig. 5., confirming the specificity of these bands to the BDNF antibody. However, other non-specific bands were detected in the secondary control lane, indicating that these bands were not specific for BDNF. To verify the results seen with the BDNF primary antibody ŽAmgen., normal ŽH77. hippocampal and temporal cortex protein samples were incubated with a

B. Connor et al.r Molecular Brain Research 49 (1997) 71–81

different polyclonal primary BDNF antibody Ždiluted 1 : 1000, N-20, Santa Cruz Biotechnology, Cat. No. sc-546.. In contrast to the Amgen antibody, this primary antibody to BDNF only detected the 14-kDa band protein ŽFig. 6.. Unfortunately, this antibody did not work on human tissue sections so we were unable to compare its pattern of immunostaining with that obtained with the other antisera ŽAmgen..

4. Discussion This study examined whether an alteration in BDNF expression occurred at the protein level in the human post-mortem Alzheimer’s disease hippocampus and temporal cortex using immunohistochemical techniques. The results of this study revealed a reduction in both the number of BDNF-immuopositive neurons and the intensity of immunostaining within cell bodies in the Alzheimer’s disease hippocampus and temporal cortex when compared to the normal human brain. The specificity of the BDNF primary antibody within the human post-mortem brain was confirmed using Western blotting techniques. Appel w3x originally proposed that Alzheimer’s disease pathology may be due to a deficit in neurotrophic factor protein or receptor expression. This proposal was extended by Hefti et al. w25x who suggested that Alzheimer’s disease pathology may be due to a reduction or alteration in the expression of the neurotrophic factor, NGF which has been reported to provide trophic support to basal forebrain cholinergic neurons w4,18,26,72,77x. However, while NGF has been proposed as a potential therapeutic agent for Alzheimer’s disease w39,40,43,59x no causal relationship between the development of Alzheimer’s disease and an alteration in NGF synthesis has been observed w1,16,31,53x. In contrast to NGF, BDNF has been observed to be widely distributed within the CNS. This observation has lead to the proposal that in addition to providing trophic support for cholinergic neurons in Alzheimer’s disease, BDNF may also promote the function and survival of other neuronal populations which are affected in Alzheimer’s disease w47,49,74x. Extending and supporting the results of previous studies w54,63x, we observed a reduction in both the number of BDNF-immunopositive neurons and the intensity of immunostaining within cell bodies in the hippocampus and temporal cortex of the human post-mortem Alzheimer’s disease brain when compared to the normal human brain. A significant reduction in the mean density of BDNF immunoreactivity was detected within the hilar region of the Alzheimer’s disease hippocampus. In addition, the results revealed that the mean number of BDNF-immunopositive cell bodies in the Alzheimer’s disease temporal cortex was greatly reduced when compared to the normal human brain. These observations are consistent with a decrease in afferent activity within the Alzheimer’s

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disease hippocampus and suggest that, within the Alzheimer’s disease brain an altered programme of gene expression may occur, resulting in aberrant levels of BDNF mRNA expression w54x and a concomitant reduction in BDNF protein as observed in the present study. While it is not possible to determine whether the reduction in BDNF protein expression precedes the development of the disease process, it is possible that the decrease in BDNF may be involved in the primary pathogenesis of Alzheimer’s disease. BDNF is a basic dimeric 28-kDa protein of non-covalently linked 14-kDa subunits w67,75x. The prepro- form of BDNF has been detected as a 28-kDa protein while the mature form of BDNF is seen at f 14 kDa w57x. Using normal hippocampal and temporal cortex protein samples, Western blotting techniques were used to verify the specificity of the primary BDNF antibody within the human post-mortem brain. In the normal hippocampal and temporal cortex samples, two BDNF-immunopositive bands were detected at f 28 and 14 kDa. These results are in agreement with previous investigations into the expression of BDNF protein in the normal rat brain w21,67,75,79x. In contrast, the BDNF primary antibody supplied by Santa Cruz Biotechnology only detected a 14-kDa band in both the normal hippocampal and temporal cortex samples. These results suggest that while the Amgen antibody may detect both the prepro- and mature forms of BDNF, the Santa Cruz antibody may detect only the mature form Ž14 kDa. of the protein. b-Amyloid protein deposition and neurofibrillary tangles are not only observed in Alzheimer’s disease but also in Down’s syndrome patients. Down’s syndrome pathology is the result of chromosome 21 trisomy and Down’s syndrome patients over the age of 30 are reported to form plaques and tangles similar to those observed within the Alzheimer’s brain w9,10,24,42,55,66,78x. b-Amyloid protein is derived from abnormal cleavage of the amyloid precursor protein ŽAPP. which is encoded by the APP gene on chromosome 21 w34,69x. Morphologically, the pathology of the plaques and tangles that occur in Alzheimer’s disease and Down’s syndrome are similar however, the mean age of pathological onset differs between the two disorders, possibly suggesting the Down’s syndrome is more closely resembles familial Alzheimer’s disease w8x. These observations provide a link between the pathogenesis of Down’s syndrome and Alzheimer’s disease. Further supporting the similarity in pathological changes between Alzheimer’s disease and Down’s syndrome, a reduction in BDNF immunoreactivity was observed in the Down’s syndrome hippocampal and temporal cortical sections comparable to that observed in the Alzheimer’s disease brain sections. The regulation of BDNF within the hippocampus occurs via neuronal activity. Zafra et al. w80x showed that a balance between the glutamatergic and GABAergic systems controls the physiological levels of BDNF mRNA

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within hippocampal neurons in vitro and in vivo. Blockade of glutamatergic neurons andror stimulation of the GABAergic system results in a reduction of BDNF mRNA levels in the hippocampus w7,80x. The cholinergic neuronal system has also been observed to be involved in the regulation of BDNF mRNA levels within the hippocampus w7,36,46x. The degeneration of both the glutamatergic and cholinergic systems are characteristic neuropathological features of Alzheimer’s disease w5,15,29,30,61,65,68x and may result in the loss of BDNF mRNA regulation. Therefore, the possibility exists that the observed reduction in BDNF expression in the Alzheimer’s disease brain may be a secondary event that occurs as a result of the loss of hippocampal glutamatergic afferents and the degeneration of the cholinergic system. Alternatively, because BDNF has been proposed to provide trophic support to the basal forebrain cholinergic system it is possible that the decrease in BDNF may contribute to the progressive atrophy of basal forebrain cholinergic neurons associated with Alzheimer’s disease w63x. Alternatively, the observed reduction of BDNF in Alzheimer’s disease hippocampal sections may be a secondary effect caused by the up-regulation of astrocytes in and around senile plaques within the Alzheimer’s disease brain. Interleukin-1 ŽIL-1. is released by reactive astrocytes associated with senile plaques and has been observed to be detrimental to the long-term survival of embryonic hippocampal neurons in culture w44x. It is possible that the accumulation of IL-1 or other cytokines may be involved in the alteration in hippocampal synaptic plasticity that occurs in Alzheimer’s disease. Furthermore, the induction of IL-1 expression in the Alzheimer’s disease hippocampus may indirectly result in the observed reduction of BDNF mRNA and protein in Alzheimer’s disease. Lapchak et al. w44x reported that BDNF mRNA was down-regulated in the rat hippocampus after systemic administration of IL-1, suggesting that the observed reduction of BDNF mRNA and protein in the Alzheimer’s disease hippocampus may be due to the induced expression of IL-1. However, in contrast, Zafra et al. w81x reported that IL-1 b had no effect on the expression of either neuronal or astrocytic BDNF mRNA. As well as having an important role in neuronal protection, BDNF may also play a physiological role in the formation of stable memories. This hypothesis is based upon recent observations showing that stimulation which produces a durable form of long-term potentiation ŽLTP., induces BDNF mRNA expression in rat dentate granule cells w19x. Furthermore, BDNF itself can produce a type of LTP in hippocampal synapses w33x. Thus, a loss of BDNF in Alzheimer’s disease may be involved in both the cause and the symptoms of this neurodegenerative disorder. In a recent study, Eide et al. w22x observed that BDNF stimulation of the full-length trkB receptor expressed in a Xenopus oocyte microinjection assay elicited a PLC-g-dependent signalling pathway. Furthermore, co-expression of

the truncated trkB receptor with the full-length receptor induced a dominant inhibitory response, preventing BDNF signal transduction. These observations may suggest that naturally occurring trkB truncated receptors function as inhibitory modulators of neurotrophic factor responsiveness. Previously, we observed w14x an increased level of trkB truncated receptor immunoreactivity in b-amyloidpositive plaques in the Alzheimer’s disease hippocampus and temporal cortex. The increased expression of the trkB truncated receptor in b-amyloid plaques in the Alzheimer’s disease brain may produce a dominant inhibitory effect on the BDNF signal transduction cascade and greatly reduce the neuroprotective actions of BDNF. Therefore, the increase in trkB truncated receptor expression in combination with the reduced neuronal expression of BDNF protein in the Alzheimer’s disease hippocampus and temporal cortex may contribute to the progressive atrophy of basal forebrain cholinergic neurons associated with Alzheimer’s disease as well as to the loss of hippocampal neuronal populations. 4.1. Conclusion The results of this study revealed a reduction in both the number of BDNF-immunpositive neurons and the intensity of immunostaining within cell bodies in the human postmortem Alzheimer’s disease hippocampus and temporal cortex. A reduction in BDNF immunoreactivity was also observed in the hippocampus and temporal cortex of a Down’s syndrome case. While additional Down’s syndrome cases are required to confirm this observation, combined with the results from the human post-mortem Alzheimer’s disease hippocampus and temporal cortex, several proposals as to the mechanismŽs. involved in the reduction of BDNF in Alzheimer’s disease can be formed. While it is as yet unclear whether the observed reduction of BDNF in Alzheimer’s disease is a primary or secondary pathological event, the loss of neuroprotection afforded by BDNF may contribute to the progressive atrophy of basal forebrain cholinergic neurons associated with Alzheimer’s disease and may result in the degeneration of hippocampal neuronal populations. Furthermore, because BDNF plays a physiological role in learning and memory formation, its loss may contribute to the cognitive deterioration characteristic of Alzheimer’s disease. Therefore, drugs aimed at enhancing BDNF activity, especially through the trkB Žfull-length. receptor might provide a treatment for both the cause and the symptoms of Alzheimer’s disease. Such ‘neuroprotective cognitive enhancers’ would provide a major boost for the treatment of Alzheimer’s disease.

Acknowledgements This research was supported by the New Zealand Neurological Foundation, The Health Research Council of

B. Connor et al.r Molecular Brain Research 49 (1997) 71–81

New Zealand, the New Zealand Lotteries Health Board and the Auckland University Research Committee. Bronwen Connor holds a Health Research Council of New Zealand Postgraduate Scholarship.

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