Name: millisecond hydrogen/deuterium-exchange mass spectrometry for the study of alpha-synuclein structural dynamics under physiological conditions
Uploaded: 2022-06-23T14:30:00
Description: Watch this Scientific Journal Video about Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions at JoVE.com

NS is funded by the University Council Diamond Jubilee Scholarship. JJP is supported by a UKRI Future Leaders Fellowship [Grant number: MR/T02223X/1].

1. Protein expression and purification of aSyn
<ol>
	<li>Prepare aSyn following a previously published report9.</li>
	<li>Dialyze into a safe storage buffer (e.g., Tris, pH 7.2 ).</li>
	<li>If required, concentrate the sample (e.g., spin filter microcentrifuge tubes using 3 kDa MWCO, 14,000 x g for approximately 10-30 min, see Table of Materials). 
	NOTE: It is advised not to concentrate excessively. The integrity of the monomer ensemble has not be...

In the present article, the following procedures are described: (1) performing peptide mapping experiments on monomeric aSyn to obtain the highest sequence coverage, (2) acquiring millisecond HDX-MS data on monomeric aSyn under physiological conditions, and (3) performing data analysis and interpretation of the resulting HDX-MS data. The provided procedures are generally simple to execute, each labeling experiment typically lasts only around 8 h for three replicates and eight timepoints, and the mapping experiment lasts ...

Parkinson&#39;s disease (PD) is a neurodegenerative illness affecting millions of people worldwide1. It is characterized by the formation of cytoplasmic inclusions known as Lewy bodies and Lewy neurites in the brain&#39;s substantia nigra pars compacta region. These cytoplasmic inclusions have been found to contain aggregates of the intrinsically disordered protein aSyn2. In PD and other synucleinopathies, aSyn transforms from a soluble disordered state into an insoluble, highly structured diseased state. In its native form, monomeric aSyn adopts a wide range of conformations stabilized by long-range electrostatic interactions between its N- and C-termini and hydrophobic interactions between its C-terminus and non-amyloid beta component (NAC) region3,4,5,6. Any disruptions in those stabilizing interactions, such as mutations, post-translational modifications, and changes in the local environment, can lead to the misfolding of the monomer, thus triggering the process of aggregation7.
While a vast amount of research exists on the oligomeric and fibrillar forms of aSyn8,9,10,11, there is a crucial need to study the monomeric form of the protein and better understand which conformers are functional (and how) and which are prone to aggregate8,9,10,11. Being intrinsically disordered, only 14 kDa in size, and difficult to crystallize, the aSyn monomer is not amenable to most structural biological techniques. However, one technique capable of measuring the conformational dynamics of monomeric aSyn is millisecond HDX-MS, which has recently generated important structural observations that would be challenging or impossible to obtain otherwise12,13,14. Millisecond HDX-MS sensitively measures the average of the protein conformational ensemble by monitoring the isotopic exchange at amide hydrogens, indicating solvent accessibility and hydrogen-bonding network participation of a particular protein region on the millisecond timescale. It is necessary to stress the millisecond aspect of the HDX-MS as, due to its natively unfolded, meta-stable nature, aSyn exhibits very fast hydrogen-exchange kinetics that manifest well below the lower limit of conventional HDX-MS systems. For example, most of the aSyn molecule has completely exchanged hydrogen for deuterium under intracellular conditions in less than 1 s. Several laboratories have now built fast-mixing instrumentation; in this case, a prototype fast-mixing quench-flow instrument capable of performing HDX-MS with a dead-time of 50 ms and a temporal resolution of 1 ms is used15. While millisecond HDX-MS has recently been acutely important in the study of aSyn, it stands to be valuable in studying intrinsically disordered proteins/regions more widely and a large number of proteins with loops/regions that are only weakly stable. For example, peptide drugs (e.g., insulin; GLP-1/glucagon; tirzepatide) and peptide-fusion proteins (e.g., the HIV inhibitor FN3-L35-T1144) are major drug formats where solution-phase structural and stability information can be a critical input for drug development decisions, and, yet, the peptide moiety is often only weakly stable and intractable by HDX-MS at the seconds timescale16,17,18,19,20. Emergent HDX-MS methods with labeling in the seconds/minutes domains have been shown to derive structural information for DNA G-quadruplexes, but it should be possible to extend this to more diverse oligonucleotide structures by the application of millisecond HDX-MS21.
HDX-MS experiments can be performed at three different levels: (1) bottom-up (whereby the labeled protein is digested proteolytically), (2) middle-down (whereby the labeled protein is digested proteolytically, and the resulting peptides are fragmented further by soft-fragmentation techniques), and (3) top-down (whereby soft-fragmentation techniques directly fragment the labeled protein)22. Thus, sub-molecular HDX-MS data allow us to localize the exchange behavior to specific regions of a protein, making it critical to have adequate sequence coverage for such experiments. The structural resolution of any HDX-MS experiment relies on the number of proteolytic peptides or fragments derived from the protein upon digestion or soft-fragmentation, respectively. In each of the three experiment types outlined above, the change in amide exchange at each peptide/fragment is mapped back onto the protein&#39;s primary structure to indicate the behavior of localized regions of the protein. While the highest structural resolution is achieved through soft-fragmentation, the description of these experiments is out of the scope of the current study, which focuses on the measurement of aSyn monomer conformations. Excellent results can be obtained with the commonly applied &#34;bottom-up&#34; workflow described here.
Here, procedures are provided on (1) how to prepare and handle aSyn samples and HDX-MS buffers, (2) how to perform peptide mapping for a bottom-up HDX-MS experiment, (3) how to acquire HDX-MS data on monomeric aSyn under physiological conditions, specifically in the millisecond time domain (using a custom-built instrument; alternative instruments for millisecond labeling have also been described), and (4) how to process and analyze the HDX-MS data. Methods using monomeric aSyn at physiological pH (7.40) in two solution conditions are exemplified here. While critically useful in the study of aSyn, these procedures can be applied to any protein and are not limited to intrinsically disordered proteins.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 &#215; 100 mm ACQUITY BEH 1.7 &#956;m C18 column&#160;</td>
			<td>Waters Corporation</td>
			<td>186002346</td>
			<td>Analytical column</td>
		</tr>
		<tr>
			<td>Acetonitrile HPLC grade &#62;99.9% HiPerSolv</td>
			<td>VWR</td>
			<td>20060.420</td>
			<td>For LC mobile phases</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma Aldrich</td>
			<td>C5670</td>
			<td>Salt for HDX buffers</td>
		</tr>
		<tr>
			<td>Chronos</td>
			<td>Axel Semrau (Purchased from Waters Corporation)</td>
			<td>667006090</td>
			<td>Scheduling software to enable multiple HDX-MS sample injections automatically. Alternative software is available from other vendors e.g. HDXDirector or LEAP Shell</td>
		</tr>
		<tr>
			<td>Deuterium chloride</td>
			<td>Goss Scientific (Cambridge Isotope Laboratories)</td>
			<td>DLM-2-50</td>
			<td>For HDX labelling buffers</td>
		</tr>
		<tr>
			<td>Deuterium oxide (99.9% D2O)</td>
			<td>Goss Scientific (Cambridge Isotope Laboratories)</td>
			<td>DLM-4</td>
			<td>Deuterated water</td>
		</tr>
		<tr>
			<td>DynamX 3.0</td>
			<td>Waters Corporation</td>
			<td>176016027</td>
			<td>Isotopic assignment and deuterium incorporation calculation</td>
		</tr>
		<tr>
			<td>Enzymate BEH Pepsin Column</td>
			<td>Waters Corporation</td>
			<td>186007233</td>
			<td>Pepsin digestion column</td>
		</tr>
		<tr>
			<td>Formic Acid, 99.0% LC/MS Grade</td>
			<td>Fisher Scientific</td>
			<td>10596814</td>
			<td>For LC mobile phases</td>
		</tr>
		<tr>
			<td>Guanidinium hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>RDD001-500G</td>
			<td>Chaotrope/Denaturant</td>
		</tr>
		<tr>
			<td>HDfleX</td>
			<td>University of Exeter</td>
			<td>N/A</td>
			<td>https://ore.exeter.ac.uk/repository/handle/10871/127982</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma Aldrich</td>
			<td>P3911</td>
			<td>Salt for HDX buffers</td>
		</tr>
		<tr>
			<td>LEAP HDX-2 CTC PAL sampling robot</td>
			<td>Waters Corporation</td>
			<td>725000637</td>
			<td>Autosampler robot</td>
		</tr>
		<tr>
			<td>Leucine enkephalin</td>
			<td>Waters Corporation</td>
			<td>186006013</td>
			<td>For mass spectrometry lockspray calibration.</td>
		</tr>
		<tr>
			<td>MassLynx</td>
			<td>Waters Corporation</td>
			<td>667004007</td>
			<td>Software controlling inlet methods and mass spectrometer</td>
		</tr>
		<tr>
			<td>Maximum recovery vials</td>
			<td>Waters Corporation</td>
			<td>600000670CV</td>
			<td>100 pack including caps - used for quench tray in LEAP HDX-2</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma Aldrich</td>
			<td>M8266</td>
			<td>Salt for HDX buffers</td>
		</tr>
		<tr>
			<td>Millipore 0.22 &#181;m syringe filters</td>
			<td>Millipore</td>
			<td>N9CA7069B</td>
			<td>Syringe filters</td>
		</tr>
		<tr>
			<td>ms2min</td>
			<td>Applied Photophysics Ltd</td>
			<td>N/A</td>
			<td>fast-mix quench-flow millisecond hdx instrument</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S9888</td>
			<td>Salt for HDX buffers</td>
		</tr>
		<tr>
			<td>Peltier temperature controller</td>
			<td>LEAP Technologies Inc.</td>
			<td>HP115-COOL/D</td>
			<td>Peltier controller to set precise temperature of chambers in the LEAP robot.</td>
		</tr>
		<tr>
			<td>ProteinLynx Global Server 3.0</td>
			<td>Waters Corporation</td>
			<td>715001030</td>
			<td>Peptide identification software. Alternative software is available from other vendors.</td>
		</tr>
		<tr>
			<td>Reagent pot caps</td>
			<td>Waters Corporation</td>
			<td>186004632</td>
			<td>100 pack</td>
		</tr>
		<tr>
			<td>Reagent pots for LEAP HDX-2</td>
			<td>Waters Corporation</td>
			<td>186001420</td>
			<td>100 pack excluding caps - used for buffers in LEAP HDX-2</td>
		</tr>
		<tr>
			<td>Sodium deuteroxide (99.5% in D2O)</td>
			<td>Goss Scientific (Cambridge Isotope Laboratories)</td>
			<td>DLM-57</td>
			<td>For HDX labelling buffers</td>
		</tr>
		<tr>
			<td>Spin filter microcentrifuge tubes (3 kDa MWCO)</td>
			<td>Amicon (Merck Sigma Aldrich)</td>
			<td>UFC5003</td>
			<td>Micro centrifuge tubes to concentrate protein. This facilitates buffer exchange and accurate sample loading for HDX-MS experiments.</td>
		</tr>
		<tr>
			<td>Synapt G2-Si mass spectrometer</td>
			<td>Waters Corporation</td>
			<td>176850035</td>
			<td>Mass spectrometer</td>
		</tr>
		<tr>
			<td>Total recovery vials</td>
			<td>Waters Corporation</td>
			<td>600000671CV</td>
			<td>100 pack including caps - used for labelling tray in LEAP HDX-2</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma Aldrich</td>
			<td>T3253-250G</td>
			<td>Buffer</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma Aldrich</td>
			<td>T60040-B2005</td>
			<td>Buffer</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma Aldrich</td>
			<td>U5378-1KG</td>
			<td>Chaotrope/Denaturant</td>
		</tr>
		<tr>
			<td>VanGuard 2.1 x 5 mm ACQUITY BEH C18 column&#160;</td>
			<td>Waters Corporation</td>
			<td>186004623</td>
			<td>Trap desalting column</td>
		</tr>
	</tbody>
</table>

millisecond hydrogen/deuterium-exchange mass spectrometry for the study of alpha-synuclein structural dynamics under physiological conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

Due to its intrinsically disordered nature, it is difficult to capture the intricate structural changes in aSyn at physiological pH. HDX-MS monitors isotopic exchange at backbone amide hydrogens, probing the protein conformational dynamics and interactions. It is one of the few techniques to acquire this information at high structural and temporal resolutions. This protocol is broadly applicable to a wide range of proteins and buffer conditions, and this is exemplified by the measurement of the exchange kinetics of aSyn ...

Watch this Scientific Journal Video about Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions at JoVE.com

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...

All organisms have disorder in their proteome, estimated up to 33%These regions are observable by hydrogen/deuterium exchange mass spectrometry, but only in the millisecond time range. Our protocol will show you how to do this successfully. This is a fully automated analytical technique.You just set everything up, program the desired time measurements, hit go and walk away. This technique opens up the exciting opportunity of screening for compounds that stabilize specific confirmations of intrinsically disordered proteins such alpha-synuclein. To begin sample preparation for peptide mapping, first take a vial of stored alpha-synuclein protein stock from the minus 80 degrees Celsius freezer.And once it is thawed, filter using a 0.22 micrometer syringe filter. Then measure the absorbance of the solution at 218 nanometers to determine the protein concentration using Beer-Lambert's law, and dilute the protein with equilibrium buffer to a final concentration of five micromolar. Next, to set up the autosampler robot, add 50 microliters of the five micromolar protein solution to a total recovery vial, and place the vial in the sample position in the right chamber of the hydrogen/deuterium exchange, or HDX, robotics in line with the mass spectrometer.Then put one vial of equilibrium buffer and two vials of pepsin wash buffer to reagent positions one, four, and five of the left chambers and one vial of quench buffer to reagent position one in the HDX right chamber and set the temperature at 20 degrees Celsius using the Peltier temperature controller. Then add an equal number of total recovery vials and maximum recovery vials in the same reaction positions of the HDX left chamber and HDX right chamber respectively. Next, set up a sample list with appropriate LC and MS methods in the scheduling software and start the schedule.To obtain the final peptide coverage map, manually curate the isotopic assignments in the DynamX HDX data analysis software. After cleaning the Fast HDX prototype instrument, open the graphical user interface of the compatible HDX software to initialize the system. Enter 20 degrees Celsius and 0.5 degrees Celsius as the sample chamber and quench chamber temperatures respectively, followed by clicking set to apply the new temperatures.To clean and prepare the instrument, set up the centrifuge tubes with LC/MS grade water at all inlets, then check both left and right syringes in the titrator plumbing delivery tab and continue clicking prime until all latent air bubbles in the tubes are gone. To check all the boxes for syringes, first go to the macros tab and click on calibrate syringes home position, then first click wash syringe load loop, followed by wash all mixing loop volumes. If there is any bubble in a buffer syringe, disconnect the syringe and degas it by vertically ejecting the bubble.Before recalibrating to the zero position, replace the syringe. For setting up the Fast HDX prototype instrument for HDX experiments, first insert the left titration inlet tube into the total recovery vial. To prevent temperature-induced oligomerization and aggregation, place the tube in a tabletop fridge.Next, add 50 milliliters of equilibrium, labeling, quench, and pepsin wash buffers to the respective buffer inlets in the right and left chambers of the instrument, followed by adding 50 milliliters of column wash buffer to the pepsin wash inlet. For priming the protein and column wash lines, check the right and left syringes in the titrator plumbing delivery tab and click prime once. After checking all the boxes for syringes as demonstrated earlier, go to the manual quench flow tab to enter the required settings.For a time course experiment, use the symbolic dot button to enter the times in milliseconds. Then set the trap time to three and the wait for HPLC to trap time plus run time plus 1.5 minutes. If blank experiments are run between sample runs, click on the run blank box after ensuring an entry in the acquisition software for the blank run after each sample run.Once the sample list is ready and the appropriate entries have been highlighted, start the run by clicking play and Fast HDX in the software. For data processing, open the file menu of the file of spectrally assigned peptides from the peptide mapping experiments and click open in the DynamX software, then import the raw files to the DynamX software. After opening the data menu, click on MS Files.To create states for each protein condition being studied, click on new state. To add each HDX time point, click on new exposure, then click new raw. To import raw files, drag each file to the correct position and click OK when finished.After automatically assigning isotopes, manually curate the isotopic assignment to ensure high data quality. Export the cluster data in csv file with the column order as described in the manuscript. Open the data menu in the mass measurement software and click on export cluster data.For data analysis, first load the exported clustered data into the HDX analysis software HDfleX. To fit the experimental data for all experiments and states, select the appropriate back exchange correction methods to get the observed rate constants for the HDX reaction. Then calculate a global significance threshold using a preferred method in HDfleX and perform hybrid significance testing to determine the significant differences across the states compared.The mapping experiment on alpha-synuclein generated a peptide coverage map that covered 100%of the protein sequence with an average redundancy of 3.79. Moreover, this protocol enabled the generation of deuterium uptake curves for different peptides of alpha-synuclein, which were then expressed as fitted and back exchange corrected graphs. It was evident that most hydrogen/deuterium exchange in alpha-synuclein was done by one second.The conformational difference between states A and B of alpha-synuclein was also evident from the higher deuterium uptake by state B both at the peptide and amino acid levels. For such a sensitive technique, it is important to use robust statistical significance tests when comparing data. Here, we used our software HDfleX.

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Research

JoVE Journal

Neuroscience

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and result in impermanent tissue levels of the drug. Additionally, drug delivery to the brain is delicate due to the blood brain barrier (BBB), thus significantly reducing intracerebral concentrations of selective drugs after systemic administration. Therefore, a system that allows both constant drug delivery without peak levels and circumvention of the BBB is in order to achieve sufficiently high intracerebral concentrations of drugs that are impermeable to the BBB. In this context, miniosmotic pumps represent an ideal system for constant drug delivery at a fixed known rate that eludes the problem of daily injection stress in animals and that may also be used for direct brain delivery of drugs. Here, we describe a method for miniosmotic pump implantation and post operatory care that should be given to animals in order to successfully apply this technique. We embed the aforementioned experimental paradigm in standard procedures that are used for studying neuroplasticity within the brain of C57BL6 mice. Thus, we exposed animals to 30 min brain infarct and implanted with miniosmotic pumps connected to the skull via a cannula in order to deliver a pro-plasticity drug. Behavioral testing was done during 30 days of treatment. After removal the animals received injections of anterograde tract tracers to analyze neuronal plasticity in the chronic phase of recovery. Results indicated that neuroprotection by the delivered drug was accompanied with increase in motor fibers crossing the midline of the brain at target structures. The results affirm the value of these techniques for drug administration and brain plasticity studies in modern neuroscience.

In order to study brain reorganization under pathological conditions we used miniosmotic pumps for direct protein delivery into the brain circumventing the blood brain barrier. Tract tracers are then injected to study alterations in brain connectivity under the influence of the protein.

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.