Brain-derived neurotrophic factor and neurotrophin-3 levels in Alzheimer's disease brains

In the present study, we investigated the levels of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in post-mortem brain tissue of Alzheimer's disease (AD) patients, and we observed a significant increase of BDNF concentration in hippocampus and parietal cortex of AD patients, as well as a negative correlation between NT-3 levels and age in hippocampus and putamen of control subjects, and for BDNF in frontal cortex. A defining feature of AD is the post-mortem identification of neuritic plaques and neurofibrillary tangles in the brain, however, a more significant neuropathological finding is the degeneration of cholinergic neurones of the basal forebrain, critically involved in memory and cognition. Neurotrophic factors are partly responsible for the maintenance of neuronal function and structural integrity in the adult brain. Our results provide, therefore, evidence that, under conditions of progressive neurodegeneration the brain stimulates the over expression of certain neurotrophic factors as a possible mechanisms of compensation, and that during senescence the expression of these molecules is regulated.     

Sodium tungstate decreases the phosphorylation of tau through GSK3 inactivation

Tungstate treatment increases the phosphorylation of glycogen synthase kinase-3beta (GSK3beta) at serine 9, which triggers its inactivation both in cultured neural cells and in vivo. GSK3 phosphorylation is dependent on the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) induced by tungstate. As a consequence of GSK3 inactivation, the phosphorylation of several GSK3-dependent sites of the microtubule-associated protein tau decreases. Tungstate reduces tau phosphorylation only in primed sequences, namely, those prephosphorylated by other kinases before GSK3beta modification, which are serines 198, 199, or 202 and threonine 231. The phosphorylation at these sites is involved in reduction of the interaction of tau with microtubules that occurs in Alzheimer's disease.     

Expression of a truncated tau protein induces oxidative stress in a rodent model of tauopathy

Truncation of tau protein and oxidative stress have been implicated as important pathogenetic events in tauopathies including Alzheimer's disease (AD). We have generated a transgenic rat model that expresses a human truncated tau protein analogous to a variant form derived from sporadic AD. We employed this model to investigate the relationship between tau protein truncation and oxidative stress. We have found that rat cortical neurons (derived from transgenic animals) that had been cultured in vitro for 16 days showed an increased accumulation of reactive oxygen species (up to 1.4-fold increase; P < 0.01) when compared to neurons derived from nontransgenic control animals. Transgene-expressing neurons treated with inducers of oxidative stress, such as glucose oxidase (GO) and buthionine sulfoximine (BSO), displayed dramatically reduced survival (31.4 +/- 3.3 and 24.9 +/- 3.6%, respectively; both P < 0.001) compared to neurons from control animals (79.9 +/- 7.1%, survival following treatment with GO and to 98.2 +/- 3.8%, survival following treatment with BSO). The number of mitochondria in processes of neurons from transgenic animals was decreased by about one-third from that present in neurons from control animals. The results reveal that expression of a human truncated variant form of tau protein leads to the accumulation of reactive oxygen species and sensitizes rat cortical neurons to cell death induced by oxidative stress. This indicates that truncation of tau may precede oxidative stress in the pathogenesis of neurodegenerative diseases such as AD and other tauopathies. These findings may have implications for therapeutic strategies aiming at prevention of neurofibrillary degeneration and cognitive decline, and identify potential new targets for drug development.     

Brain-derived neurotrophic factor triggers a rapid glutamate release through increase of intracellular Ca(2+) and Na(+) in cultured cerebellar neurons

We reported previously that BDNF induced glutamate release was dependent on intracellular Ca(2+) but not extracellular Ca(2+) in cerebellar neurons (Numakawa et al., 1999). It was revealed that the release was through a non-exocytotic pathway (Takei et al., 1998; Numakawa et al., 1999). In the present study, we monitored the dynamics of intracellular Ca(2+) and Na(+) in cerebellar neurons, and investigated the possibility of reverse transport of glutamate mediated by BDNF. As reported, BDNF increased the intracellular Ca(2+) level. We found that the Ca(2+) increase induced by BDNF was completely blocked by xestospongin C, an IP(3) receptor antagonist, and U-73122, a PLC-gamma inhibitor. Xestospongin C and U-73122 also blocked the BDNF-dependent glutamate release, suggesting that the BDNF-induced transient increase of Ca(2+) through the activation of the PLC-gamma/ IP(3) pathway was essential for the glutamate release. We found that BDNF induced a Na(+) influx. This was blocked by treatment with TTX. U-73122 and xestospongin C blocked the BDNF-induced Na(+) influx, suggesting that the Na(+)influx required the BDNF-induced Ca(2+) increase. Next, we examined the possibility that a co-transporter of Na(+) and glutamate was involved in the BDNF-induced glutamate release. BDNF-induced glutamate release was blocked by L-trans-pyrollidine-2,4-dicalboxylic acid (t-PDC), a glutamate transporter inhibitor, whereas neither the 4-aminopyridine (4AP)- nor high potassium (HK(+))-induced release was blocked by t-PDC. In addition, DL-threo-beta-benzyloxyaspartate (DL-TBOA) also blocked the BDNF-mediated glutamate release, suggesting that reverse transport of glutamate may be involved. All the results therefore suggest that Na(+)-dependent reverse transport contributes to BDNF-mediated transmitter release through the PLC-gamma/IP(3)-mediated Ca(2+) signaling.     

Expression of reticulon 3 in Alzheimer's disease brain

Aims: Reticulon 3 (RTN3), a member of the reticulon family of proteins, interacts with the β-secretase, β-site amyloid precursor protein-cleaving enzyme 1 (BACE1), and inhibits its activity to produce β-amyloid protein. The aim of the present study was to clarify the biological role of RTN3 in the brain and its potential involvement in the neuropathology of Alzheimer's disease (AD). Methods: We performed immunohistochemical and biochemical analyses using a specific antibody against RTN3 to investigate the expression and subcellular localization of RTN3 in control and AD brain tissue samples. Results: Western blot analysis revealed no significant differences in the RTN3 levels between control and AD brains. Immunohistochemical staining showed that RTN3 immunoreactivity was predominantly localized in pyramidal neurones of the cerebral cortex. The patterns of RTN3 immunostaining were similar in control and AD cerebral cortices, and senile plaques were generally negative for RTN3. Biochemical subcellular fractionation disclosed that RTN3 colocalized with BACE1 in various fractions, including the endoplasmic reticulum and the Golgi apparatus. Double-immunofluorescence staining additionally indicated that RTN3 was localized in both endoplasmic reticulum and Golgi compartments in neurones. Conclusions: These results show that RTN3 is primarily expressed in pyramidal neurones of the human cerebral cortex and that no clear difference of RTN3 immunoreactivity is observable between control and AD brains. Our data also suggest that there is considerable colocalization of RTN3 with BACE1 at a subcellular level.     

Site-specific effects of tau phosphorylation on its microtubule assembly activity and self-aggregation

Microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles in brains with Alzheimer's disease. The phosphorylation sites of tau are mainly localized in the proline-rich (residues 172–251) and C-terminal tail (residues 368–441) regions, which flank the microtubule-binding repeats. Here, we investigated the effects of tau phosphorylation at these distinct sites/regions on its activity of stimulating microtubule assembly and its self-aggregation. We found that tau phosphorylation at the proline-rich region by dual-specificity tyrosine-phosphorylated and -regulated kinase 1A inhibited its microtubule assembly activity moderately and promoted its self-aggregation slightly. Tau phosphorylation at the C-terminal tail region by glycogen synthase kinase-3β increased its activity and promoted its self-aggregation markedly. Tau phosphorylation at both regions plus the microtubule-binding region by cAMP-dependent protein kinase diminished its activity (∼70% inhibition) and disrupted microtubules. These studies reveal the differential regulation of tau's biological activity and self-aggregation by phosphorylation at various sites/regions.     

GSK3β overexpression induces neuronal death and a depletion of the neurogenic niches in the dentate gyrus

Overexpression of GSK3β in transgenic mice induces learning deficits and some features associated with Alzheimer's disease (AD), including dentate gyrus (DG) atrophy. Here, we assessed whether these mice also recapitulate DG atrophy as well as impaired neurogenesis reported in AD. Ultrastructural analysis revealed that there were fewer and more disorganized neurogenic niches in these animals, coupled with an increase in the proportion of immature neurons. Indeed, the maturation of granule cells is delayed as witnessed by the alterations to the length and patterning of their dendritic trees and to the mossy fiber terminals. Together with an increase in neuronal death, these phenomena lead to a marked decrease in the number and disorganization of granule cells of the DG. Our results suggest that GSK3β overexpression perturbs proliferation and maturation, resulting in the loss of immature neurons. In turn, the activation of microglia is stimulated in conjunction with a decrease in the birth of new functional neurons, leading to the deterioration of this structure. These data support the idea that by inducing degeneration of the DG, GSK3β could be involved in the pathogenesis of AD. © 2010 Wiley‐Liss, Inc.     

Regulation of tau phosphorylation and protection against beta-amyloid-induced neurodegeneration by lithium. Possible implications for Alzheimer's disease

Alzheimer's disease is a neurodegenerative disorder characterized by the accumulation of the β-amyloid peptide and the hyperphosphorylation of the tau protein, among other features. The most widely accepted hypothesis on the etiopathogenesis of this disease proposes that the aggregates of the β-amyloid peptide are the main triggers of tau hyperphosphorylation and the subsequent degeneration of affected neurons. In support of this view, fibrillar aggregates of synthetic β-amyloid peptide induce tau hyperphosphorylation and cell death in cultured neurons. We have previously reported that lithium inhibits tau hyperphosphorylation and also significantly protects cultured neurons from cell death triggered by β-amyloid peptide. As lithium is a relatively specific inhibitor of glycogen synthase kinase-3 (in comparison with other protein kinases), and other studies also point to a relevant role of this enzyme, we favor the view that glycogen synthase kinase-3 is a crucial element in the pathogenesis of Alzheimer's disease. In our opinion, the possibility of using lithium, or other inhibitors of glycogen synthase kinase-3, in experimental trials aimed to ameliorate neurodegeneration in Alzheimer's disease should be considered.     

C677T polymorphism of methylenetetrahydrofolate reductase gene affects plasma homocysteine level and is a genetic factor of late-onset Alzheimer's disease

Background: Elevated plasma homocysteine levels are known as a risk for atherosclerotic vascular disease and venous thrombosis and have been shown as a risk for late‐onset Alzheimer's disease (LOAD). Method: To examine the effect of genetic factors predisposing to elevated plasma homocysteine levels on the occurrence of LOAD, we determined the genotype of a C677T polymorphism of methylenetetrahydrofolate reductase (MTHFR) gene and a variable number tandem repeat (VNTR) spanning exon 13–intron 13 boundary of cystathionine β‐synthase (CBS) gene in patients with LOAD and community‐based control subjects. Results: Logistic regression indicated that the MTHFR‐T allele was a risk for LOAD (P < 0.05), independently from apolipoprotein E‐?4 (APOE‐?4) allele. Kaplan–Meier tests showed that in APOE‐?4 non‐carriers, individuals with the MTHFR‐TT genotype have occurences of LOAD earlier than those with the MTHFR‐CC genotype (P < 0.05). Multiple regression analysis indicates that MTHFR‐T allele increases plasma homocysteine levels (P = 0.0002), while the number of X chromosomes decreases (P = 0.01). Plasma homocysteine level was not correlated with age, plasma albumin reflecting nutritional condition, and the dose of APOE‐?4 allele. The CBS‐20 VNTR allele showed the same trend to increase plasma homocysteine level as the MTHFR‐T allele, but a risk effect for LOAD was not evident. Conclusion: A genetic propensity for elevated plasma homocysteine levels, explained by the MTHFR‐T allele encoding defective enzymatic function, is involved in the development of LOAD, particularly in APOE‐?4 non‐carriers, and that homocysteine metabolism could be a preventive target to LOAD in the elderly.     

Increased level of active GSK-3beta in Alzheimer's disease and accumulation in argyrophilic grains and in neurones at different stages of neurofibrillary degeneration

The somatodendritic accumulation of hyperphosphorylated tau proteins is an early event preceding the appearance of neurofibrillary tangles (NFT) in Alzheimer's disease (AD) and might be necessary for their formation. Glycogen synthase kinase-3beta (GSK-3beta) is a physiological kinase for tau that generates many tau phosphorylation sites identified in NFT and in other tau-positive inclusions. We have studied the cellular distribution and the expression of the active form of GSK-3beta (GSK-3 pTyr216) in AD patients, in argyrophilic grain disease and in diffuse Lewy body disease. By Western blotting analysis, a significant increase in the level of GSK-3 (pTyr216) was observed in the frontal cortex of AD patients. A population of neurones showed a somatodendritic accumulation of GSK-3 (pTyr216) but not of the inactive form of GSK-3beta (GSK-3 pSer9). Most of these GSK-3 (pTyr216)-positive cells were positive for six different phosphotau epitopes known to be generated by GSK-3beta. By using a quadruple labelling method using GSK-3 (pTyr216) and phosphotau immunolabelling combined with Gallyas and DAPI staining, we examined neurones containing a somatodendritic GSK-3 (pTyr216) immunoreactivity at different stages of neurodegeneration. A majority of neurones at the pretangle stage without Gallyas-positive inclusions were GSK-3 (pTyr216) positive and this GSK-3 (pTyr216) immunoreactivity remained in most cells containing Gallyas and phosphotau-positive inclusions excepted in extracellular NFT. A GSK-3 (pTyr216) immunoreactivity was present in argyrophilic grains but not in cortical Lewy bodies. These results directly suggest that the activity of GSK-3beta is increased in AD and that somatodendritic accumulation and activation of GSK-3beta is an early event preceding and accompanying the formation of NFT and of other tau-positive inclusions.     

A transitory activation of protein kinase-A induces a sustained tau hyperphosphorylation at multiple sites in N2a cells-imply a new mechanism in Alzheimer pathology

Summary. Overactivation of protein kinase in the end stage of Alzheimer’s disease brain has not been established. The purpose of the present study was to explore the possible mechanism for protein kinases in leading to Alzheimer-like tau hyperphosphorylation. We found that incubation of N2a/tau441 with forskolin, a specific activator of cAMP-dependent protein kinase (PKA), induced an increased phosphorylation level of tau at both PKA and non-PKA sites in a dose- and time-dependent manner, and the hyperphosphorylation of tau was positively correlated with the elevation of PKA activity. When the cells were transitorily incubated with forskolin, a temporary activation of PKA with a sustained and almost equally graded tau hyperphosphorylation at some non-PKA sites was observed. In either case, the activity of glycogen synthase kinase-3 (GSK-3) was not changed. It is suggested that only transitory activation of PKA in early stage of Alzheimer disease may result in a sustained tau hyperphosphorylation at multiple sites, implying a new mechanism to Alzheimer-like tau hyperphosphorylation. The first and second authors contributed equally to the paper     

Intranasal NAP administration reduces accumulation of amyloid peptide and tau hyperphosphorylation in a transgenic mouse model of Alzheimer's disease at early pathological stage

Accumulation of β-amyloid (Aβ) peptide and hyperphosphorylation of tau in the brain are pathological hallmarks of Alzheimer's disease (AD). Agents altering these pathological events might modify clinical disease progression. NAP (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) is an octapeptide that has shown neuroprotective effects in various in vitro and in vivo neurodegenerative models. Previous studies showed that NAP protected against Aβ-induced neurotoxicity, inhibited Aβ aggregation, and, by binding to tubulin, prevented disruption of microtubules. In this study, we investigated the effect of NAP on Aβ and tau pathology using a transgenic mouse model that recapitulates both aspects of AD. We administered NAP intranasally (0.5 μg/mouse per day, daily from Monday through Friday) for 3 mo, starting from 9 mo of age, which is a prepathological stage in these mice. NAP treatment significantly lowered levels of Aβ-1–40 and 1–42 in brain. In addition, NAP significantly reduced levels of hyperphosphorylated tau. Of particular interest, hyperphosphorylation at the threonine 231 site was reduced; phosphorylation at this site influences microtubule binding. Our results indicate that NAP treatment of transgenic mice initiated at an early stage reduced both Aβ and tau pathology, suggesting that NAP might be a potential therapeutic agent for AD.     

Role of tau in the polymerization of peptides from β-amyloid precursor protein

The composition of paired helical filaments (PHFs), the intracellular amyloid fibrils that accumulate in the brains of Alzheimer patients, is not completely known. We investigated whether synthetic peptides from β-amyloid precursor protein (APP) can form PHF-like fibrils. Two peptides formed fibrils morphologically similar to PHFs. The presence of tau protein, a known PHF component, greatly enhanced the numbers of fibrils formed from one peptide, from the C-terminus of APP, and became associated with the fibrils. A τ fragment corresponding to the tubulin-binding region was sufficient to induce fibril formation. Tau did not alter fibril formation by the other peptide, which was from the β/A4 region of APP. These results raise the possibility that a C-terminal fragment of APP, along with tau, may be involved in PHF formation. Thus the proteolytic processing of APP may generate fragments that contribute to both amyloids and both histopathologic lesions of Alzheimer's disease.     

Modulation of brain-derived neurotrophic factor as a potential neuroprotective mechanism of action of omega-3 fatty acids in a parkinsonian animal model

While we recently reported the beneficial effects of omega-3 polyunsaturated fatty acids (n−3 PUFAs) in a mouse model of Parkinson's disease (PD), the mechanisms of action remain largely unknown. Here, we specifically investigated the contribution of the brain-derived neurotrophic factor (BDNF) to the neuroprotective effect of n−3 PUFA observed in a mouse model of PD generated by a subacute exposure to MPTP using a total of 7 doses of 20 mg/kg over 5 days. The ten-month high n−3 PUFA treatment which preceded the MPTP exposure induced an increase of BDNF mRNA expression in the striatum, but not in the motor cortex of animals fed the high n−3 PUFA diet. In contrast, n−3 PUFA treatment increased BDNF protein levels in the motor cortex of MPTP-treated mice, an effect not observed in vehicle-treated mice. The mRNA expression of the high-affinity BDNF receptor tropomyosin-related kinase B (TrkB) was increased in the striatum of MPTP-treated mice fed the high n−3 PUFA diet compared to vehicle and MPTP-treated mice on the control diet and to vehicle mice on the high n−3 PUFA diet. These data suggest that the modulation of BDNF expression contributes, in part, to n−3 PUFA-induced neuroprotection in an animal model of PD.     

Distribution of Alzheimer''s disease amyloid protein precursor in normal human and rat nervous system

Alzheimer's disease and cerebral amyloid angiopathies (CAA) are clinically heterogeneous diseases, but pathogenically related by the deposition of beta A4-amyloid in the brain in the form of neuritic plaques and/or vascular infiltrates. Antibodies directed against the N-terminal region of the predicted sequence of the beta A4 amyloid protein precursor (APP) were used to investigate the cellular distribution of this protein in the brain of normal humans and rats. We found a widespread presence of APP throughout the nervous tissue, including neurons, blood vessels, meningeal membranes, choroid plexus and ependymal cells. The highest APP immunoreactivity in both species was found in neuronal cell bodies and their processes, and around blood vessels. These findings may account for the clinical, pathological and aetiological differences found among the beta A4-amyloidosis.     

Transgenic mice overexpressing the neurotrophic factor S-100β show neuronal cytoskeletal and behavioral signs of altered aging processes: implications for Alzheimer's disease and Down's syndrome

S-100β is a neurotrophic factor released by astroglial cells and localized to chromosome 21, within the region which is considered obligate for Down's syndrome (DS). S-100β is increased in the postmortem brains of both DS and Alzheimer's disease. Transgenic mice, produced by insertion of the human gene for S-100β, were examined for dendritic development at two ages, using an antibody against microtubule associated protein-2 (MAP-2). At the earliest stages, the density of dendrites within the hippocampus of transgenic animals exceeded that of controls. Also, MAP-2 immunostaining was evident in the region of the cell body. By 1 year of age, the transgenic animals had significant loss of dendrites compared to controls and the number of cells showing cell body staining was further increased. These pathological changes could be indicative of the presence of neurofibrillary tangles and cytoskeletal collapse. Behaviorally, younger transgenic animals could not perform in a learning task as well as controls. Together, these findings suggest that increased S-100β in brain may lead to accelerated development, followed by increased aging. The pathological changes may prove useful as an animal model of Down's syndrome and Alzheimer's disease.