in vitro assessment of aggregated amyloid-&beta; on neuronal growth cone collapse

In Vitro Assessment of Aggregated Amyloid-&beta; on Neuronal Growth Cone Collapse

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-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-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 mostly 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 4 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 3 or 4 as axons. Axonal growth cones lacking lamellipodia or possessing fewer than three filopodia are considered collapsed growth cones.

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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) and incubate them at 37 &#176;C overnight.</li>
		<li>Remove the PDL solution and wash the wells 3 times with distilled water.</li>
	</ol>
	</li>
	<li>Neuron culture
	<ol>
		<li>Mince freshly isolated cerebral cortices from embryonic day 14 (E14) ddY (Deutschland, Denken, and Yoken) mice with microscissors in neuron culture medium containing 12% horse serum, 0.6% glucose, and 2 mM L-glutamine (medium A). Do not add antibiotics. 
		NOTE: In this protocol, the ddY mouse is used. This is an outbred strain commonly used in Japan. This neuron culture protocol can be also applied for rat cortical neurons.</li>
		<li>Centrifuge the tissues at 87 x g for 3 min.</li>
		<li>Remove the supernatant. Then to the pellet, add 2 mL of 0.05% trypsin and incubate for 15 min at 37&#176;C. Mix by tapping every 5 min.</li>
		<li>Add 4 mL of medium A and mix by tapping.</li>
		<li>Centrifuge the tissues at 178 x g for 3 min.</li>
		<li>Remove the supernatant, and incubate the tissues with 600 U/mL deoxyribonuclease I (DNase I) and 0.3 mg/mL soybean trypsin inhibitor dissolved in PBS for 15 min at 37 &#176;C. Mix by tapping every 5 min.</li>
		<li>After incubation, add 4 mL of medium A and mix by tapping.</li>
		<li>Centrifuge the tissues at 178 x g for 3 min.</li>
		<li>After removing the supernatant, add 4 mL of medium A and triturate the tissues with a polished Pasteur pipette.</li>
		<li>Filter the triturated tissues with a 70 &#181;m pore-size mesh. After filtration, calculate the density of cells with a hemocytometer.</li>
		<li>Culture the cells in the 8-well culture slide at 0.8 x 104 cells/well with medium A and maintain them in a CO2 incubator with a humidified atmosphere of 10% CO2 at 37 &#176;C.</li>
		<li>After 4 h of culturing, replace the culture medium to one containing 2% supplement for neuronal culture, 0.6% glucose, and 2 mM L-Glutamine (medium B). 
		NOTE: The purity of neurons was approximately 75%, as described previously.</li>
	</ol>
	</li>
	<li>Collapse assay
	<ol>
		<li>Dissolve commercially obtained full-length amyloid &#946;1-42 (A&#946;1-42) in distilled water at a concentration of 0.5 mM and incubate at 37 &#176;C for 7 days. After the incubation, store the aggregated A&#946;1-42 solution in a -30 &#176;C freezer until use. 
		NOTE: This incubation is necessary for aggregation and toxicity of A&#946;.</li>
		<li>After 4 days of neuronal culture, treat the wells with 100 &#956;L of new medium B, containing 0.5 &#956;M aggregated A&#946;1-42 or vehicle solution (distilled water) for 1 h. 
		NOTE: Effects of A&#946;1-42 were dose-dependently increased from 0.1 to 5 &#956;M and peaked at 0.5 &#956;M as described previously. Similar results can be observed when by A&#946;1-42 treatment for 1 h after 3 days of neuronal culture.</li>
		<li>Remove the culture medium and immediately fix the neurons with 4% paraformaldehyde containing 4% sucrose in PBS for 1 h at 37 &#176;C on a hot plate.</li>
		<li>After fixation, wash the neurons 3 times with PBS and mount them with an aqueous mounting medium. Dry the mounting medium at 4 &#176;C for 2&#8211;4 days.</li>
		<li>Capture the entire area (7.8 x 9 mm2) of each well with a 20X dry objective lens on an inverted microscope.</li>
		<li>Classify the longest neurites of each neuron in stage 3 or 4 as axons, as previously described.</li>
		<li>Classify growth cones according to the following criteria: 1) axonal growth cones lacking lamellipodia or 2) possessing fewer than three filopodia are considered collapsed growth cones, as described previously. 
		NOTE: Healthy growth cones are scored as 0 point; collapsed growth cones are scored as 1 point. Mean collapse scores are calculated for each treatment.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ddY mice</td>
			<td>SLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eight-well culture slide</td>
			<td>Falcon</td>
			<td>354108</td>
			<td></td>
		</tr>
		<tr>
			<td>poly D lysine</td>
			<td>Wako</td>
			<td>168-19041</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture medium, Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>house serum</td>
			<td>Gibco</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Wako</td>
			<td>049-31165</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Wako</td>
			<td>074-00522</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% trypsin</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Worthington</td>
			<td>DP</td>
			<td></td>
		</tr>
		<tr>
			<td>soybean trypsin inhibitor</td>
			<td>Gibco</td>
			<td>17075-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter with 70 &#181;m mesh size, cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Astec</td>
			<td>SCA-165DS</td>
			<td></td>
		</tr>
		<tr>
			<td>Amyloid &#946;1-42</td>
			<td>Sigma-Aldrich</td>
			<td>A9810</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Wako</td>
			<td>162-16065</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Wako</td>
			<td>196-00015</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqueous mounting medium, AquaPoly/Mount</td>
			<td>polysciences</td>
			<td>18606-20</td>
			<td></td>
		</tr>
		<tr>
			<td>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>
			<td>Objective Plan-Apochromat 20x</td>
			<td>Carl Zeiss</td>
			<td>420650-9901</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Kuboyama, T. <a href="https://app.jove.com/v/58229">Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons.</a> J. Vis. Exp. (2018)
This video demonstrates an in vitro assessment of neuronal growth cone collapse by amyloid-&#946; (A&#946;) aggregates. Treating the neurons with A&#946; aggregates causes cytoskeletal destabilization, leading to the collapse of growth cones at the tip of the axons. The treated cells are compared with control cells to assess the extent of growth cone collapse.

1. Collapse Assay

	Poly-D-lysine coating 
	
		Coat 8-well culture slides with 400 &#956;L of 5 &#956;g/mL poly-D-lysine (PDL) in phosphate-buffered ...

Begin with a multi-well culture slide containing neurons from a mouse embryo in a medium.
The neurons exhibit growth cones at the tips of their growing axons. These structures include thin projections called filopodia and sheet-like extensions called lamellopodia, which consist of actin filaments and microtubules, components of the cytoskeleton.
Treat the test wells with aggregated amyloid-&#946;, or A&#946;, protein and the control wells with a vehicle solution.
In the test wells, aggregated A&#946; binds to membrane receptors, initiating downstream signaling that phosphorylates cytoskeleton-regulating proteins.
The phosphorylated proteins disrupt actin filaments and microtubules, destabilizing the growth cone&#39;s structural integrity and causing its collapse.
Incubate with a fixative to preserve the cellular structure, then wash the cells and mount them for imaging.
Using a microscope, compare the cellular structures in the test and control wells.
An increased occurrence of growth cone collapse in A&#946;-treated neurons indicates the toxic effect of A&#946; aggregates.

25 vistas. Evaluación in vitro de la β amiloide agregada en el colapso del cono de crecimiento neuronal

Video: Evaluación in vitro de la β amiloide agregada en el colapso del cono de crecimiento neuronal - Experimento

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

Análisis de anomalías inducida por β-amiloide en la estructura del coágulo de fibrina por espectroscopía y microscopía electrónica de barrido

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

Investigación

JoVE Journal

Bioquímica

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

In situ Visualización del crecimiento de axones y la dinámica de los conos de crecimiento en cultivos agudos de cortes cerebrales embrionarios ex vivo

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

Neurociencias

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

Análisis de la dinámica de la actina, el acoplamiento del embrague y la fuerza de tracción para el avance del cono de crecimiento

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...

In Vitro Assessment of Aggregated Amyloid-β on Neuronal Growth Cone Collapse

Transcripción

In Vitro Assessment of Aggregated Amyloid-β on Neuronal Growth Cone Collapse