<meta charset="utf8"></head><body>Primer Hipokampal Nöronlardaki Proteinlerin Canlı Hücre Görüntüleme ile Görselleştirilmesi

<ol>
	<li>Kneynsberg, A., Combs, B., Christensen, K., Morfini, G., Kanaan, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+degeneration+in+tauopathies:+disease+relevance+and+underlying+mechanisms.">Axonal degeneration in tauopathies: disease relevance and underlying mechanisms.</a> Front Neurosci. 11, 572 (2017).</li><li>Combs, B., Mueller, R. L., Morfini, G., Brady, S. T., Kanaan, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+and+axonal+transport+misregulation+in+tauopathies.">Tau and axonal transport misregulation in tauopathies.</a> Adv Exp Med Biol. 1184, 81-95 (2019).</li><li>Brady, S. T., Morfini, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+motor+proteins,+axonal+transport+deficits+and+adult-onset+neurodegenerative+diseases.">Regulation of motor proteins, axonal transport deficits and adult-onset neurodegenerative diseases.</a> Neurobiol Dis. 105, 273-282 (2017).</li><li>Morfini, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+defects+in+neurodegenerative+diseases.">Axonal transport defects in neurodegenerative diseases.</a> J Neurosci. 29 (41), 12776-12786 (2009).</li><li>Brady, S. T., Lasek, R. J., Allen, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+axonal+transport+in+extruded+axoplasm+from+squid+giant+axon.">Fast axonal transport in extruded axoplasm from squid giant axon.</a> Science. 218 (4577), 1129-1131 (1982).</li><li>LaPointe, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amino+terminus+of+tau+inhibits+kinesin-dependent+axonal+transport:+implications+for+filament+toxicity.">The amino terminus of tau inhibits kinesin-dependent axonal transport: implications for filament toxicity.</a> J Neurosci Res. 87 (2), 440-451 (2009).</li><li>Kanaan, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+forms+of+tau+inhibit+kinesin-dependent+axonal+transport+through+a+mechanism+involving+activation+of+axonal+phosphotransferases.">Pathogenic forms of tau inhibit kinesin-dependent axonal transport through a mechanism involving activation of axonal phosphotransferases.</a> J Neurosci. 31 (27), 9858-9868 (2011).</li><li>Kanaan, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+in+the+amino+terminus+of+tau+prevents+inhibition+of+anterograde+axonal+transport.">Phosphorylation in the amino terminus of tau prevents inhibition of anterograde axonal transport.</a> Neurobiol Aging. 33 (4), 826.e15-826.e30 (2012).</li><li>Pigino, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+fast+axonal+transport+is+a+pathogenic+mechanism+for+intraneuronal+amyloid+beta.">Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta.</a> Proc Natl Acad Sci U S A. 106 (14), 5907-5912 (2009).</li><li>Morfini, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+huntingtin+inhibits+fast+axonal+transport+by+activating+JNK3+and+phosphorylating+kinesin.">Pathogenic huntingtin inhibits fast axonal transport by activating JNK3 and phosphorylating kinesin.</a> Nat Neurosci. 12 (7), 864-871 (2009).</li><li>Morfini, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+fast+axonal+transport+by+pathogenic+SOD1+involves+activation+of+p38+MAP+kinase.">Inhibition of fast axonal transport by pathogenic SOD1 involves activation of p38 MAP kinase.</a> PLoS One. 8 (6), e65235 (2013).</li><li>Combs, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frontotemporal+lobar+dementia+mutant+tau+impairs+axonal+transport+through+a+protein+phosphatase+1gamma-dependent+mechanism.">Frontotemporal lobar dementia mutant tau impairs axonal transport through a protein phosphatase 1gamma-dependent mechanism.</a> J Neurosci. 41 (45), 9431-9451 (2021).</li><li>Christensen, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphomimetics+at+Ser199/Ser202/Thr205+in+tau+impairs+axonal+transport+in+rat+hippocampal+neurons.">Phosphomimetics at Ser199/Ser202/Thr205 in tau impairs axonal transport in rat hippocampal neurons.</a> Mol Neurobiol. 60 (6), 3423-3438 (2023).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat Protoc. 1 (5), 2406-2415 (2006).</li><li>Seibenhener, M. L., Wooten, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+hippocampal+neurons+from+prenatal+mice.">Isolation and culture of hippocampal neurons from prenatal mice.</a> J Vis Exp. (65), 3634 (2012).</li><li>Neumann, S., Chassefeyre, R., Campbell, G. E., Encalada, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KymoAnalyzer:+a+software+tool+for+the+quantitative+analysis+of+intracellular+transport+in+neurons.">KymoAnalyzer: a software tool for the quantitative analysis of intracellular transport in neurons.</a> Traffic. 18 (1), 71-88 (2017).</li><li>Cox, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+isoform-specific+tau+aggregates+suggests+a+common+toxic+mechanism+involving+similar+pathological+conformations+and+axonal+transport+inhibition.">Analysis of isoform-specific tau aggregates suggests a common toxic mechanism involving similar pathological conformations and axonal transport inhibition.</a> Neurobiol Aging. 47, 113-126 (2016).</li><li>Tiernan, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudophosphorylation+of+tau+at+S422+enhances+SDS-stable+dimer+formation+and+impairs+both+anterograde+and+retrograde+fast+axonal+transport.">Pseudophosphorylation of tau at S422 enhances SDS-stable dimer formation and impairs both anterograde and retrograde fast axonal transport.</a> Exp Neurol. 283 (Pt A), 318-329 (2016).</li><li>Mueller, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau:+a+signaling+hub+protein.">Tau: a signaling hub protein.</a> Front Mol Neurosci. 14, 647054 (2021).</li><li>Hedstrom, K. L., Ogawa, Y., Rasband, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AnkyrinG+is+required+for+maintenance+of+the+axon+initial+segment+and+neuronal+polarity.">AnkyrinG is required for maintenance of the axon initial segment and neuronal polarity.</a> J Cell Biol. 183 (4), 635-640 (2008).</li><li>Basu, H., Schwarz, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QuoVadoPro,+an+autonomous+tool+for+measuring+intracellular+dynamics+using+temporal+variance.">QuoVadoPro, an autonomous tool for measuring intracellular dynamics using temporal variance.</a> Curr Protoc Cell Biol. 87 (1), e108 (2020).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4% Trypan blue</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 mL microcentrifuge tubes</td>
			<td>DOT</td>
			<td>RN1700-GMT</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe with 21 G needle</td>
			<td>Fisher</td>
			<td>14-826-84</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL plastic syringe</td>
			<td>Fisher</td>
			<td>14-823-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>14 G needle</td>
			<td>Fisher</td>
			<td>14-817-203</td>
			<td></td>
		</tr>
		<tr>
			<td>15 G needle</td>
			<td>Medline</td>
			<td>SWD200029Z</td>
			<td></td>
		</tr>
		<tr>
			<td>16 G needle</td>
			<td>Fisher</td>
			<td>14-817-104</td>
			<td></td>
		</tr>
		<tr>
			<td>18 G needle</td>
			<td>Fisher</td>
			<td>14-840-97</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G needle</td>
			<td>Fisher</td>
			<td>14-840-90</td>
			<td></td>
		</tr>
		<tr>
			<td>32% paraformaldehyde</td>
			<td>Fisher</td>
			<td>50-980-495</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 647 goat anti-rabbit IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A21244</td>
			<td>RRID:AB_2535813</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco</td>
			<td>15290-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Arruga Micro Embryonic Capsule Forceps, Curved; 4&#34;&#160;</td>
			<td>Roboz</td>
			<td>RS-5163</td>
			<td>autoclave</td>
		</tr>
		<tr>
			<td>B-27 Supplement (50x), serum free</td>
			<td>Gibco</td>
			<td>A3582801</td>
			<td></td>
		</tr>
		<tr>
			<td>BioCoat 24-well Poly D lysine plates&#160;</td>
			<td>Fisher</td>
			<td>08-774-124</td>
			<td></td>
		</tr>
		<tr>
			<td>boric acid</td>
			<td>Sigma</td>
			<td>B6768-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo 3 1/2&#34; Long 8 x 0.15 mm Angle Sharp Scissors</td>
			<td>Roboz</td>
			<td>RS-5658</td>
			<td>autoclave</td>
		</tr>
		<tr>
			<td>Cell counting device</td>
			<td></td>
			<td></td>
			<td>automatic or manual</td>
		</tr>
		<tr>
			<td>Confocal microscope with live cell chamber attachment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal imaging software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-glucose</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I (Worthington)</td>
			<td>Fisher</td>
			<td>NC9185812</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>14200-075</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Fisher</td>
			<td>O2783-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fatal-Plus Solution</td>
			<td>Vortech Pharmaceuticals, LTD</td>
			<td>NDC 0298-9373-68</td>
			<td>sodium pentobarbital; other approved methods of euthanasia may be used&#160;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin Reagent Solution</td>
			<td>Gibco</td>
			<td>15710-072</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>glutamine substitute</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution</td>
			<td>Gibco</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ version 1.51n</td>
			<td>ImageJ</td>
			<td></td>
			<td>Life-Line version 2017 May 30: https://imagej.net/software/fiji/downloads</td>
		</tr>
		<tr>
			<td>KymoAnalyzer (version 1.01)</td>
			<td>Encalada Lab</td>
			<td></td>
			<td>Package includes all 6 macros: https://www.encalada.scripps.edu/kymoanalyzer</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000</td>
			<td>Invitrogen</td>
			<td>100022050</td>
			<td>Use with P3000 transfection enhancer reagent</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Fisher</td>
			<td>AC223211000</td>
			<td></td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>Sigma</td>
			<td>M8250</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors 3.5&#34; Straight Sharp/Sharp</td>
			<td>Roboz</td>
			<td>RS-5910</td>
			<td>autoclave</td>
		</tr>
		<tr>
			<td>Neurobasal Plus medium</td>
			<td>Gibco</td>
			<td>A3582901</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurofascin (A12/18) Mouse IgG2a</td>
			<td>UC Davis/NIH NeuroMab</td>
			<td>75-172</td>
			<td>RRID:AB_2282826; 250 ng/mL; Works in rat neurons, NOT in mouse neurons</td>
		</tr>
		<tr>
			<td>Neurofascin 186 (D6G60) Rabbit IgG</td>
			<td>Cell Signaling</td>
			<td>15034</td>
			<td>RRID:AB_2773024; 500 ng/mL; Works in mouse neurons, we have not tested in rat neurons</td>
		</tr>
		<tr>
			<td>newborn calf serum</td>
			<td>Gibco</td>
			<td>16010-167</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31985-062</td>
			<td></td>
		</tr>
		<tr>
			<td>P3000</td>
			<td>Invitrogen</td>
			<td>100022057</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, 100 x 10 mm glass</td>
			<td>Fisher</td>
			<td>08-748B</td>
			<td>For dissection; autoclave</td>
		</tr>
		<tr>
			<td>Petri dish, 100 x 20 mm glass</td>
			<td>Fisher</td>
			<td>08-748D</td>
			<td>To place uterine horns in; autoclave</td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P7886-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene conical centrifuge tubes (15 mL)</td>
			<td>Fisher</td>
			<td>14-955-238</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene conical centrifuge tubes (50 mL)</td>
			<td>Fisher</td>
			<td>14-955-238</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Fisher</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma</td>
			<td>S5636</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium borate decahydrate</td>
			<td>VWR</td>
			<td>MK745706</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight-Blade Operating Scissors Blunt/Sharp</td>
			<td>Fisher</td>
			<td>13-810-2</td>
			<td>autoclave</td>
		</tr>
		<tr>
			<td>Syringe Filters, 0.22 &#181;m</td>
			<td>VWR</td>
			<td>514-1263</td>
			<td></td>
		</tr>
		<tr>
			<td>Thumb dressing forceps, serrated, 4.5&#34;</td>
			<td>Roboz</td>
			<td>RS-8100</td>
			<td>autoclave</td>
		</tr>
		<tr>
			<td>&#181;-Slide 4 Well Glass Bottom</td>
			<td>Ibidi</td>
			<td>80427</td>
			<td></td>
		</tr>
	</tbody>
</table>

using live&#45;cell imaging to measure the effects of pathological proteins on axonal transport in primary hippocampal neurons

Neurons depend on the bidirectional transport of cargo along the axon to maintain functional synapses and neural connectivity. Axonal transport deficits are thought to be critical contributors to the pathogenesis of several neurodegenerative diseases, including Alzheimer&#39;s disease (AD) and other tauopathies, Parkinson&#39;s disease, amyotrophic lateral sclerosis, and Huntington&#39;s disease1,2,3. Indeed, pathological modifications to several disease-related proteins negatively affect transport (reviewed in 4). Developing methods to investigate the mechanisms by which pathological proteins exert toxicity in disease is necessary to understand neurodegenerative disorders and identify potential targets for therapeutic intervention.
Several important insights into axon transport, including the discovery of conventional kinesin, the kinase- and phosphatase-dependent pathways regulating motor proteins, and mechanisms by which pathological proteins disrupt the regulation of axon transport, were made using the isolated squid axoplasm model4,5. Perfusion of squid axoplasm with pathological forms of tau protein inhibits anterograde fast axonal transport (FAT), an effect dependent on the exposure of the phosphatase-activating domain of tau, which activates protein phosphatase 1 (PP1)6,7,8. PP1 activates glycogen synthase kinase 3 (GSK3), which in turn phosphorylates kinesin light chains causing the release of cargo. Another pathological protein in AD is amyloid-&#946;. Oligomeric forms of amyloid-&#946; inhibit bidirectional FAT through casein kinase 2, which phosphorylates kinesin light chains9. Furthermore, pathological huntingtin protein harboring a polyglutamine expansion and a familial ALS-linked SOD1 mutant each disrupt axonal transport in the squid axoplasm through c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activity, respectively10,11.
While the squid axoplasm model continues to be a valuable tool in understanding the effects of pathological proteins on axonal transport, limited access to the equipment and specimens prevents it from being more widely used. We developed a transport assay using live-cell confocal microscopy imaging of rodent (mouse and rat) primary neurons. This model represents an easily adaptable and manipulatable mammalian neuron-based approach using widely available cell sources and microscopy systems. For example, a variety of pathological proteins (e.g., harboring disease-related modifications) are expressed to identify how specific modifications of these proteins affect transport in axons. Similarly, a variety of fluorescently-tagged cargo proteins can be used to examine cargo-specific changes. Moreover, the underlying molecular mechanisms are studied relatively easily by targeting expression (i.e., knockdown or overexpression) of selected proteins that may mediate these effects. This method also is easily adapted for primary neurons derived from a wide variety of animal models.
We present a detailed protocol describing the live-cell axon transport assay that was previously used in primary hippocampal neurons to show that mutant tau proteins associated with frontotemporal lobar dementias (FTLD; P301L or R5L tau) increase the pausing frequency of fluorescently-labeled cargo proteins&#160;bidirectionally12. Furthermore, the knockdown of the PP1&#947; isoform rescued the pausing effects12. This provides support for the model of pathological tau-induced disruption that is mediated through aberrant activation of a signaling pathway initiated by PP1 as described above6,7,12. In a separate study, we showed that pseudophosphorylation of tau at S199/S202/T205 (the pathogenic AT8 phosphoepitope relevant to tauopathies) increased cargo pause frequency and anterograde segment velocity. These effects were dependent on the N-terminal phosphatase activating domain of tau13. These examples highlight the utility of this model for identifying the mechanisms of how pathological proteins disrupt axon transport in mammalian neurons.
This paper provides a detailed description of the method beginning with the harvest, dissociation, and culturing of primary mouse hippocampal neurons, followed by the transfection of the neurons with cargo proteins fused with a fluorescent protein, and finally, the live-cell imaging and image analysis approach. We demonstrate how this method is used to study the effects of modified tau on the bidirectional transport of the vesicle-associated protein, synaptophysin, as an example. However, there is flexibility in the pathogenic protein and transport cargo protein of interest, which makes this a versatile approach to study axonal transport.

<meta charset="utf8"></head><body>Yazar Spotlight: Nörodejeneratif Hastalıklarda Patolojik Proteinlerin Aksonal Taşıma Üzerindeki Etkisini Anlamak

Primer hipokampal nöronlarda patolojik proteinlerin aksonal taşıma üzerindeki etkilerini ölçmek için canlı hücre görüntülemenin kullanılması

Read this Scientific Article Protocol about Author Spotlight: Understanding the Impact of Pathological Proteins on Axonal Transport in Neurodegenerative Diseases at JoVE.com

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal ...

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Neuroscience

Using Live&#45;Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

305 Görüntüleme.  Michigan State University. Nöronlar, fonksiyonel sinapsis ve nöral bağlantıyı sürdürmek için akson boyunca yükün çift yönlü taşınmasına güvenir. Taşımadaki eksikliklerin, çeşitli nörodejeneratif hastalıkların patogenezine kritik katkıda bulunduğu düşünülmektedir. Tau proteininde hastalıkla ilişkili modifikasyonların aksonal transportu bozduğu mekanizmaları tanımlamayı amaçlıyoruz.Tau proteininin çoklu patolojik formlarının memeli nöronl...

Video: Yazar Spotlight: Nörodejeneratif Hastalıklarda Patolojik Proteinlerin Aksonal Taşıma Üzerindeki Etkisini Anlamak

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-&#946;, &#945;-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions.
Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellular&#160;domain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport.
Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

Here, we demonstrate how to combine transfection of primary hippocampal rodent neurons with live-cell confocal imaging to analyze pathological protein-induced effects on axonal transport and identify mechanistic pathways mediating these effects.