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 author has nothing to disclose.

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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		<tr height="40">
			<td height="40" style="height:40px;width:339px;">ddY mice</td>
			<td style="width:133px;">SLC</td>
			<td style="width:109px;"></td>
			<td style="width:539px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Eight-well culture slide</td>
			<td>Falcon</td>
			<td>354108</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">poly D lysine</td>
			<td>Wako</td>
			<td>168-19041</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Culture medium, Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">house serum</td>
			<td>Gibco</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">glucose</td>
			<td>Wako</td>
			<td>049-31165</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-glutamine</td>
			<td>Wako</td>
			<td>074-00522</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.05% trypsin</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNase I</td>
			<td>Worthington</td>
			<td>DP</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">soybean trypsin inhibitor</td>
			<td>Gibco</td>
			<td>17075-029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Filter with 70 &#181;m mesh size, cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">B-27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CO2 incubator</td>
			<td>Astec</td>
			<td>SCA-165DS</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Amyloid &#946;1-42</td>
			<td>Sigma-Aldrich</td>
			<td>A9810</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">paraformaldehyde</td>
			<td>Wako</td>
			<td>162-16065</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">sucrose&#160;</td>
			<td>Wako</td>
			<td>196-00015</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Aqueous mounting medium, Aqua-Poly/Mount</td>
			<td>polysciences</td>
			<td>18606-20</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Inverted microscope A</td>
			<td>Carl Zeiss</td>
			<td>Axio Observer Z1&#160;</td>
			<td>Connected with AxioCam MRm, Heating Unit XL S, CO2 Module S1, and TempModule S1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective Plan-Apochromat 20x</td>
			<td>Carl Zeiss</td>
			<td>420650-9901</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective Plan-Apochromat 63x</td>
			<td>Carl Zeiss</td>
			<td>440762-9904</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective, CFI Plan Apo Lambda 40X</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anti-MAP2 IgG</td>
			<td>Abcam</td>
			<td>ab32454</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anti-tau-1 IgG</td>
			<td>Chemicon</td>
			<td>MAB3420</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anti-amyloid &#946; antibody</td>
			<td>IBL</td>
			<td>10379</td>
			<td>clone 11A1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">normal goat serum</td>
			<td>Wako</td>
			<td>143-06561</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">bovine serum albumin</td>
			<td>Wako</td>
			<td>010-25783</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">t-octylphenoxypolyethoxyethanol</td>
			<td>Wako</td>
			<td>169-21105</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">goat anti-mouse IgG conjugated with AlexaFluor 594</td>
			<td>Invitrogen</td>
			<td>A11032</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">goat anti-rabbit IgG conjugated with AlexaFluor 488</td>
			<td>Invitrogen</td>
			<td>A11029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">hot plate</td>
			<td>NISSIN</td>
			<td>NHP-M30N</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">cover glass</td>
			<td>Fisher Scientific</td>
			<td>12-545-85</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">35 mm dish</td>
			<td>IWAKI</td>
			<td>1000-035</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Silicone RTV</td>
			<td>Shin-Etsu</td>
			<td>KE42T</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">hand punch</td>
			<td style="width:133px;">Roper Whitney</td>
			<td style="width:109px;">No. XX</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fluorescence membrane probe, FM1-43FX</td>
			<td>Invitrogen</td>
			<td>F35355</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ca2+- and Mg2+-free Hanks&#39; balanced salt solution</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Transfection solution, Nucleofector solution</td>
			<td>Lonza</td>
			<td>VPG-1001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electroporator, Nucleofector I</td>
			<td>Amaxa</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Inverted microscope B</td>
			<td>Keyence</td>
			<td>BZ-X710</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">Image software, ImageJ</td>
			<td style="width:133px;">NIH</td>
			<td style="width:109px;"></td>
			<td style="width:539px;">https://imagej.nih.gov/ij/</td>
		</tr>
	</tbody>
</table>

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 at JoVE.com

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

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 ...

This method can help answer key questions in the field of Alzheimer disease research, especially the early events involved in growth cone collapse. The main advantage of this technique is to make it possible to visualize and analyze axonal growth cones immediately after amyloid beta treatment. Start by using micro-scissors to mince freshly isolated cerebral cortices from embryonic day-14 mice in neuron culture medium without antibiotics.Collect the tissues and centrifuge the tissues at 87G for three minutes. Following centrifugation, remove the supernatant and add two milliliters of 0.05%trypsin to the pellet. Incubate for 15 minutes at 37 degrees Celsius and mix by tapping every five minutes.After 15 minutes, add four milliliters of medium A to neutralize the trypsin, and mix by tapping. Then centrifuge the tissues at 178 times G for three minutes. After removing the supernatant, add DNAse and soybean trypsin inhibitor solution and incubate for 15 minutes at 37 degrees Celsius with mixing every five minutes as before.When the incubation time is elapsed, add four milliliters of medium A.Mix and centrifuge as before. After removing the supernatant, add four milliliters of medium A and triturate the tissues with a polished Pasteur pipette until no debris is observed. Next, filter the triturated tissues with 70-micron pore-sized mesh.After filtration, count the cells with a hemocytometer and calculate the cell density. Pipette 0.8 times 10 to the fourth cells in medium A into each well of an eight-well culture slide. Then incubate in a humidified atmosphere of 10%CO2 at 37 degrees Celsius.After four hours of culturing, change the culture medium to medium B.The purity of neurons using this method is approximately 75%Begin this assay at least a week in advance by opening a fresh vial of commercially obtained full-length A beta 42 and dissolving the contents in sterile, distilled water to a concentration of 0.5 millimolar. Incubate at 37 degrees Celsius for seven days to induce aggregation and toxicity. Prepare aliquots of aggregated A beta 1-42 and store at minus 80 degrees Celsius until use.On day four of the neuronal culture, treat the wells with 100 microliters of 0.5 micromolar aggregated A beta 1-42 or vehicle solution in medium B for one hour as previously demonstrated. After the hour has elapsed, remove the culture medium and immediately fix the neurons with 4%paraformaldehyde containing 4%sucrose in PBS for one hour at 37 degrees Celsius on a hot plate. Fixation is most important as maintaining the shape of growth cones is critical for this protocol.After fixation, wash the neurons three times with PBS. Then after removing the chamber, mount the neurons with an aqueous mounting medium. Dry the mounting medium at four degrees Celsius for two to four days.Capture the entire area of each well with a 20X dry objective lens on an inverted microscope. Capturing and analyzing the entire area in each well is important for avoiding subjectivity. Classify the longest neurites of each neuron in stage three or four as axons.Axonal growth cones lacking lamellipodia or possessing fewer than three filopodia are considered collapsed growth cones. A beta 1-42 oligomers, shown here before incubation, aggregate after seven days of incubation at 37 degrees Celsius. After treatment with aggregated A beta 1-42, immunostaining with an antibody for the toxic oligomer of A beta shows positive staining in red on A beta 1-42 treated neurons, but not vehicle treated neurons.The early phenomena induced by A beta 1-42 treatment were analyzed after four days in culture. Identified axons were confirmed as such by positive immunostaining for the axonal marker tau-1 shown in red and negative immunostaining for the dendritic marker, MAP-2, shown in green. After one hour of vehicle treatment, growth cones had spread lamellipodia and several filopodia.These were identified as healthy growth cones. Conversely, one hour of A beta 1-42 treatment led to shrunken growth cones, which developed no lamellipodia or filopodia. These were identified as collapsed growth cones.While attempting this procedure, it's important to remember to fix the cells with 4%PFA and 4%sucrose at 37 degrees Celsius to my-nor-idge this fixation protocol in the best. Following this protocol, other method like live-cell imaging and gene transfection can be performed to answer additional questions like when and how the axon growth cones are collapsed and how the collapsed growth cones are recovered.

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JoVE Journal

Neuroscience

7.9K visualizações.  University of Toyama. Este método pode ajudar a responder a perguntas-chave no campo da pesquisa da doença de Alzheimer, especialmente os primeiros eventos envolvidos no colapso do cone de crescimento. A principal vantagem dessa técnica é possibilitar visualizar e analisar cones de crescimento axonal imediatamente após o tratamento beta amiloide. Comece usando micro-tesouras para picar cortices cerebrais recém-isolados de camundongos embrionários no dia 14 em meio de cultura de neurônios sem antibióticos....