Name: visualizing axonal growth cone collapse and early amyloid &#946; effects in cultured mouse neurons
Uploaded: 2018-10-30T14:00:00
Description: Watch this Scientific Journal Video about Visualizing Axonal Growth Cone Collapse and Early Amyloid Î² Effects in Cultured Mouse Neurons Video at JoVE.com

This work was partially supported by research grants from JSPS (KAKENHI 18K07389), Japan, Takeda Science Foundation, Japan, and Kobayashi Pharmaceutical Co., Ltd., Japan.

All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals at the Sugitani Campus of the University of Toyama and were approved by the Committee for Animal Care and Use of Laboratory Animals at the Sugitani Campus of the University of Toyama (A2014INM-1, A2017INM-1).
1. Collapse Assay
<ol>
	<li>Poly-D-lysine coating
	<ol>
		<li>Coat 8-well culture slides with 400 &#956;L of 5 &#956;g/mL poly-D-lysine (PDL) in phosphate-buffered saline (PBS) an...

The protocol described in this study enabled the observation of early phenomena in axonal growth cones after A&#946;1-42 treatment. A&#946;1-42 induced endocytosis in axonal growth cones within 20 min, and growth cone collapse was observed within 1 h of treatment. This endocytosis was probably mediated by clathrin. By using this protocol, the inhibition of clathrin-mediated endocytosis was confirmed to prevent A&#946;1-42-induced growth cone collapse and axonal degeneration in cultured neurons27. ...

Amyloid-&#946; (A&#946;) deposits are found in the brain of patients with Alzheimer's disease (AD) and are considered a critical cause of AD1 that disrupt neuronal networks, leading to memory impairments2,3,4. Many clinical drug candidates have been shown to effectively prevent amyloid-&#946; (A&#946;) production or remove A&#946; deposits. However, none have succeeded in improving memory function in AD patients5. A&#946; is already deposited in the brain prior to the onset of memory impairments6; therefore, decreasing A&#946; levels in the brains of patients exhibiting memory impairments may be ineffective. A&#946; deposition is present in preclinical AD patients; however, these patients rarely present with neuronal degeneration and memory deficits6. There is a time lag between A&#946; deposition and memory impairments. Therefore, a critical strategy for the prevention of AD is blocking A&#946; toxicity signaling during the early stages of AD, prior to the development of memory deficits. A&#946; deposition induces axon degeneration7,8,9,10,11,12,13, which may lead to a disruption of neural networks and permanent impairment of memory function. Many studies have investigated the mechanisms of A&#946; toxicity; for example, the degenerated axons of AD mice brains have been shown to have increased autophagy14. Calcineurin activation has been reported as a possible mechanism of A&#946;-induced axonal degeneration15; however, the direct trigger of axonal degeneration remains unknown. 
This study focuses on the collapse of axonal endings called growth cones. The collapse of axonal growth cones can be caused by axonal growth repellents, such as semaphorin-3A and ephrin-A516,17,18,19,20. Collapse-like dystrophic axonal endings have been observed in the brains of AD patients21,22. Additionally, a failure of growth cone functioning can provoke axonal degeneration23. However, it is unknown whether A&#946; induces growth cone collapse. Therefore, this study presents a novel protocol to observe the early effects of A&#946; in cultured neurons and investigate A&#946;-induced growth cone collapse.

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			<td>10379</td>
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			<td height="40" style="height:40px;">normal goat serum</td>
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			<td height="40" style="height:40px;">bovine serum albumin</td>
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visualizing axonal growth cone collapse and early amyloid &#946; effects in cultured mouse neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the brains of AD patients, these do not improve memory functions. Since A&#946; aggregates in the brain before the appearance of memory impairments, targeting A&#946; may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to A&#946; deposition should be blocked before AD development. A&#946; induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of A&#946; toxicity, the source of A&#946; toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by A&#946; in axonal growth cones of cultured neurons. This protocol revealed that A&#946; induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents A&#946; toxicity. This protocol will be useful in studying the early effects of A&#946; and may lead to more efficient and preventative AD treatment.

In this protocol, A&#946;1-42 was incubated at 37 &#176;C for 7 days before use, because incubation of A&#946;1-42 was needed for producing toxic forms27,28,30,35. After this incubation, aggregated forms of A&#946; were observed (Figure 1A). It has been reported that similar incubation of A&#946;1-42 produced the fibril form of A&#9...

Watch this Scientific Journal Video about Visualizing Axonal Growth Cone Collapse and Early Amyloid Î² Effects in Cultured Mouse Neurons Video at JoVE.com

Visualizing Axonal Growth Cone Collapse and Early Amyloid Î² Effects in Cultured Mouse Neurons Video (Video) | JoVE

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented. This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's disease.

Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the ...

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