Neurofibrillary tangles

It has been demonstrated that neuorgenesis driven by neural precursor cells persists well into the adult period. This study was to observe the effects of Amyloid-beta (25-35) peptide (Aβ^sub 25-35^) on neurogenesis in the subventricular zone and dentate gyrus of adult mouse brain. Aggregated Aβ^sub 25-35^ (1 mg/ml, 3 µl) was injected into the lateral ventricle of adult mouse. Animals were transcardially perfused with 4% paraformaldehyde in PBS, respectively at 5, 10, 20, 30 days after the Aβ^sub 25-35^ injection. All the animals were injected with BrdU (50 mg/kg, i.p) to label the neural precursor cells 24 h before the each perfusion. NeuN immunoffuorescence and BrdU immunohistology were performed. It was found that Aβ^sub 25-35^ could injure the mature neurons and decrease the number of NeuN positive neurons. It also showed that Aβ^sub 25-35^ inhibited neurogenesis and significantly decreased the number of BrdU positive cells in the dentate gyrus of hippocampus, but it had no obvious effects on neurogenesis in the subventricular zone. The present results indicated that Aβ^sub 25-35^ could impair neurogenesis in the hippocampus of adult mouse brain. [Neurol Res 2005; 27: 218-222]

Keywords: Aβ^sub 25-35^; neurogenesis; neural precursor cells; proliferation; subventricular zone; dentate gyrus

INTRODUCTION

Neurogenesis is the proliferation of neural precursor cells (NPCs), and the migration and differentiation of new neural cells. In the adult mammal brain, neurogenesis occurs in two limited areas: the subventricular zone and dentate gyrus of the hippocampus. During the past decade, the field of neurogenesis research has been the highlight of life science. As undifferentiated cells, NPCs can proliferate and multi-differentiate in vitro and in vivo. NPCs have been found not only in the embryo and developing brain, but also in the mature brain of adult mammals although their number decreases with age1. It has been demonstrated that transplanted NPCs can survive, and differentiate into functional neurons in vivo, and repair or correct the injured function2-4. Meanwhile, endogenous NPCs can also functionally incorporate into the existing circuitry and play a physical role5. It has been concluded that NPCs are a novel disease treatment.

Alzheimer's disease (AD) is a complicated neurodegenerative disease, and the most common cause of dementia. The pathologic features include the loss of neurons and synapses, neurofibrillary tangles and senile plaque derived from the deposit of Amyloid-beta peptide (Aβ). Many researchers have revealed the pivotal roles of Aβ in the proceeds of AD6-7. At the same time, new neural cells born from NPCs could function, establish synapse connection with neurons and replace the lost neurons. So, it is interesting to explore the biological behavior of endogenous NPCs during the progress of AD, and the effects of Aβ on the NPCs.

This study investigated the effects of Aβ^sub 25-35^, the toxic peptide fragment of Aβ, on the neurogenesis driven by NPCs in the brain of adult mouse.

MATERIALS AND METHODS

Animals and reagents

Male adult BALB/c mice, (4 weeks old, 22-24 g), were provided by the Institute of Animal Sciences, Chinese Academy of Medical Sciences. They were maintained five per cage with food and water ad libitum under controlled temperature and humidity conditions with a standardized light and dark cycle (lights on at 8 a.m. and off at 8 p.m.). 5-bromo-2'-deoxyuridine (BrdU) and monoclonal mouse anti-BrdU antibody were purchased from Sigma (USA). Rodamine labeled secondary antibody was purchased from Santa Cruz Inc (USA). Monoclonal mouse anti-NeuN was purchased from Chemicon (USA).

Preparation and injection of Aβ^sub 25-35^

Aβ^sub 25-35^ was dissolved with sterile normal saline water into a concentration of 1 mg/ml, and then incubated at 37°C for 72 h to aggregate8-9. Every mouse was injected i.c.v with aggregated Aβ^sub 25-35^ 3 µl.

BrdU administration

Animals were perfused at designated times. Before each perfusion, BrdU was administrated i.p. 50 mg/kg to all animals three times every 8 h to label the endogenous neural precursor cells10.

24 h after the last BrdU adminstration, animals were transcardially perfused with 4% paraformaldehyde in PBS respectively 5, 10, 20, 30 days after the Aβ^sub 25-35^ injection. Brains were removed and postfixed overnight in the same 4% paraformaldehyde solution at 4°C, after which they were equilibrated in sucrose (30% in PBS), cut into 40 µm coronal sections on a freezing microtome.

Immunofluorescence for NeuN

Brain slices rinsed three times with TBS (tris-buffered-saline: 0.1 mol/L Tris-HCl /0.9% NaCl, pH 7.4) were then blocked with 10% goat serum, 0.3% Triton X-100 in TBS (blocking buffer) for 1 h11. Slices were then incubated with mouse monoclonal antibody to NeuN overnight at 4 C. Rodamine labeled goat anti-mouse secondary antibody was employed for 2 h at room temperature.

Immunohistology for BrdU

DNA denaturation of sections processed for BrdU immunodetection was carried out by (1) incubating sections in 50% formamide/2 × SSC at 65°C for 2 h followed by a wash in 2 × SSC for 5 min, and (2) incubating sections in 2N HCl at 37°C for 30 min. Sections were neutralized with 0.1 mol/L borate buffer (pH 8.5) for 15 min. Sections were then washed in TBS, and incubated in blocking buffer for 1 h, and then transferred to blocking buffer containing mouse monoclonal antibody to BrdU10,12-14. Sections were incubated overnight at 4°C and washed three times in TBS. Secondary antibodies were diluted in blocking buffer and applied for 2 h at room temperature. Sections were treated with horseradish for 1 h, and then developed with DAB. Negative control sections received identical preparations for immunohistochemistry staining, except for the primary antibody, which was omitted.

Count of BrdU positive cells

The number of BrdU-positive cells in the subventricular zone and in the dentate gyrus (including hilus and subgranular zone) was quantified using unbiased stereological methods12-14. BrdU-positive cells were counted in a one-in-sixth series (240 µm apart) using a 40 × objective and imaged in a cooled CCD camera.

Statistical analysis

Data were expressed as mean+ SD. ANOVA was used for comparison among groups. Values of P

RESULTS

NeuN immunofluorescence

NeuN is a specific marker of mature neurons and is often used to evaluate the number of neural cells. It was found that Aβ^sub 25-35^ damaged the mature neurons and decreased the number of NeuN positive neural cells in the hippocampus, special in the dentate gyrus (Figure 1 A-D).

Neurogenesis in vivo is studied with BrdU incorporation

BrdU is the analog of Thymidine, and could be incorporated into DNA during the S phase of the cell cycle. The result showed that BrdU positive neural precursor cells exist in the subventricular zone and the dentate gyrus. Some neural precursor cells exist in clusters and cell morphology is different in the subventricular zone (Figure 2 A-B). New cells could migrate to the dentate gyrus and differentiate into NeuN positive neurons (Figure 3 A-C).

Effect of Aβ^sub 25-35^ on the neurogenesis

It was shown that Aβ^sub 25-35^ could significantly disrupt the neurogenesis driven by neural precursor cells in the dentate gyrus of the hippocampus. BrdU positive cells were obviously decreased, significantly compared with the control (Table 1). In contrast, it had no obvious effect on the proliferation of neural precursor cells in the subventricular zone (Figure 4 A-D, data not shown).

DISCUSSION

It has been a dogma that neurogenesis in mammals only exists in the developing brain, and that the matured neurons cannot be replaced or renewed after injury or loss in the adult brain. Recently, it was found that NPCs persist well into the adult brain, and not only in developing brain. Neurogenesis in vivo included three steps: proliferation of NPC, migration and differentiation of neurons arising from the NPC15. It is regulated by many states, such as mental and physiological activity, enriched environment, stress, drugs and pathological injury, etc16-19.

It had been demonstrated that new neurons arising from the NPCs could incorporate into the neural circuitry, establish synaptic connections and play a physical function, especially in terms of hippocampusdependent learning and memory5,16. It means that neurogenesis has a functional consequence. There is considerable evidence explaining the toxicity of Aβ on the matured neurons. The results in the present study showed that Aβ^sub 25-35^ inhibited neurogenesis. It suggests a role of NPCs in the pathogenesis of AD, and provides new data of Aβ toxicity.

We found that NPCs in SVZ and in the hippocampus show different biological reactions to Aβ^sub 25-35^ toxicity. It has been reported that neural stem cells only exist in the subventricular zone, and neural progenitor cells exist in the subgranular zone of dentate gyrus in the hippocampus20. Moreover, NPCs in the SVZ can be assigned into a few types21. The inhibiting action of Aβ^sub 25-35^ is on neurogenesis in the hippocampus lasted about 20 days. We speculated that it was related to the metabolism and clearance of Aβ in vivo. In AD, the deposit of Aβ is a lasting and chronic process, so its action may be persisting. Because the hippocampus was very closely related to learning and memory, we speculated that the disruption of neurogenesis was involved in the cognitive injury of AD patients: for the decrease of proliferation and survival of NPCs, there were too few new neurons to replace the degenerated neural cells.

In conclusion, our results indicate that Aβ inhibited neurogenesis in the adult brain. It provides a novel idea, e.g. protecting or stimulating neurogenesis in vivo, to treat AD and other neurodegenerative disorders. However, further research is required to determine how and to what extent Aβ effects neurogenesis in vivo.

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Xuekun Li and Pingping Zuo

Department of Pharmacology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China

Correspondence to: Dr. ZUO Pingping, MD, PhD, Department of Pharmacology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5# Dong Dan San Tiao, Beijing 100005, China. [zuopp@public3.bta.net.cn, xklipumc@ hotmail.com]

Copyright Maney Publishing Mar 2005
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