Enrichment of single neurons and defined brain regions from human brain tissue samples for...

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T R A N S L A T I O N A L N E U R O S C I E N C E S - O R I G I N A L A R T I C L E

Enrichment of single neurons and defined brain regionsfrom human brain tissue samples for subsequent proteome

analysis

Mariana Molina2,6 • Simone Steinbach1 • Young Mok Park4,5 • Su Yeong Yun4,5 •

Ana Tereza Di Lorenzo Alho2,7 • Helmut Heinsen8 • Lea. T. Grinberg2,3,9 •

Katrin Marcus1 • Renata E. Paraizo Leite2,3 • Caroline May1

Received: 25 November 2014 / Accepted: 11 June 2015 / Published online: 30 June 2015 Springer-Verlag Wien 2015

Abstract Brain function in normal aging and neurologi-

cal diseases has long been a subject of interest. With cur-rent technology, it is possible to go beyond descriptive

analyses to characterize brain cell populations at the

molecular level. However, the brain comprises over 100

billion highly specialized cells, and it is a challenge to

discriminate different cell groups for analyses. Isolating

intact neurons is not feasible with traditional methods, such

as tissue homogenization techniques. The advent of laser

microdissection techniques promises to overcome previous

limitations in the isolation of specific cells. Here, we pro-

vide a detailed protocol for isolating and analyzing neurons

from postmortem human brain tissue samples. We describe

a workflow for successfully freezing, sectioning and

staining tissue for laser microdissection. This protocol was

validated by mass spectrometric analysis. Isolated neurons

can also be employed for western blotting or PCR. This

protocol will enable further examinations of brain cell-

specific molecular pathways and aid in elucidating distinct

brain functions.

Keywords Neurons Brain Laser microdissection

Substantia nigra

Introduction

Neurodegenerative diseases cause selective vulnerability of

specific cell populations (Mattson and Magnus 2006).

Molecular analysis of single populations of affected cells

(e.g. neurons) will lead to a better understanding of disease

mechanisms and may therefore enable the identification of targets for early-onset diagnostics and disease treatment

(Liao et al. 2004). However, the heterogeneity and com-

plexity of brain tissue make it challenging to isolate

specific brain cells. Neurons are not easily detached from

their parenchyma, are highly branched and interact in

networks. Recently, laser microdissection (LMD) hasM. Molina, S. Steinbach, R. E. P. Leite and C. May contributed

equally.

& Katrin Marcus

[email protected]

& Caroline May

[email protected]

1 Medizinisches Proteom-Center, Ruhr-Universität Bochum,

ZKF, Universitätsstraße 150, 44801 Bochum, Germany

2 Physiopathology in Aging Lab/Brazilian Aging Brain Study

Group-LIM22, University of Sao Paulo Medical School,

São Paulo, Brazil

3 Discipline of Geriatrics, University of Sao Paulo Medical

School, São Paulo, Brazil

4 Center for Cognition and Sociality, Institute for Basic

Science, Ochang, Korea

5 Mass Spectrometry Research Center, Ochang, Korea

6 Discipline of Pathophysiology, University of Sao Paulo

Medical School, São Paulo, Brazil

7 Instituto do Cérebro, Hospital Israelita Albert Einstein,

São Paulo, Brazil

8 Bavarian Julius-Maximilians-Universität, Würzburg,

Germany

9 Department of Neurology, Memory and Aging Center,

University of California, San Francisco, USA

1 3

J Neural Transm (2015) 122:993–1005

DOI 10.1007/s00702-015-1414-4

http://crossmark.crossref.org/dialog/?doi=10.1007/s00702-015-1414-4&domain=pdfhttp://crossmark.crossref.org/dialog/?doi=10.1007/s00702-015-1414-4&domain=pdf


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emerged as a promising alternative for isolating neurons

for genomics and transcriptomics (Boone et al. 2013;

Kumar et al. 2013; Majer et al. 2012; Moulédous et al.

2003; Friedrich et al. 2012; Decarlo et al. 2011; Simunovic

et al. 2009; Cantuti-Castelvetri et al. 2007; Elstner et al.

2011). For example, Simunovic et al. (2009) made a gene

expression profile of dopamine neurons isolated by LMD

for a comparison study of idiopathic PD and control subjects. This allowed for identification of different genes

associated with PD, such as, e.g. PARK. Alternatively,

LMD can also be a promising alternative for proteomic

studies. Using this technique, the tissue is first cut into thin

sections and mounted onto glass slides. With the aid of a

microscope, the cells or regions of interest are dissected

with a laser and collected in a tube (Fig. 1). An interme-

diate staining step between tissue mounting and dissection

enables cells to be distinguished by molecular features

rather than by morphological characteristics. Furthermore,

even smaller structures, such as inclusion bodies, can be

isolated for analyses (Hashimoto et al. 2012; Minjarez et al.2013). Despite the numerous advantages offered by LMD,

this method has yet to be broadly incorporated into neu-

roscience proteomic studies due to technical limitations. In

this study, using the human substantia nigra as an example,

we provide a detailed workflow for human brain studies

using LMD that overcomes technical limitations for pro-

teomic analysis. This approach can be used for highly

specific studies of the neuronal proteome, facilitating the

further understanding of complex brain function and cir-

cuitry. Specifically, LMD enables the researcher to char-

acterize proteome changes, which would be masked in

global analyses, making LMD an essential tool for neu-

ronal proteomic studies.

Methods

Ethical statement

All the protocols were approved by the ethics committee of

the University of São Paulo and the Ruhr-University

Bochum, as well as the Brazilian federal research ethics

committee. Informed written consent was obtained from

the next of kin.

Subjects

Postmortem human brains of elderly subjects were supplied

by the Brain Bank of the Brazilian Brain Aging Study

Group (BBBABSG) at the University of Sao Paulo Medical

School (USPMS). The BBBABSG collects brains fromdeceased individuals aged C50 years. The methods used

by the BBBABSG have been previously reported (Grinberg

et al. 2007).

Defining the optimal anatomical plane for neuron

collection

Two brains of control human subjects were fixed by

immersion in 10 % formalin for at least 4 weeks. There-

after, the brainstem was severed by a horizontal cut ventral

to the superior colliculi. The formalin-fixed brainstems

were dehydrated in a graded series of ethanol solutions,

embedded in celloidin, and serially sectioned at 350 lm

thickness in either the horizontal or the sagittal plane

(Heinsen et al. 2000; Theofilas et al. 2014). The sections

were stained with gallocyanin (Nissl staining) and mounted

as previously described in detail (Heinsen et al. 2000). The

cell arrangement and density of cells were compared in the

substantia nigra in each section.

Freezing for laser microdissection

Three midbrains were collected. Each midbrain was

mediosagittally sectioned, resulting in three right and three

left half-midbrains. Each one of the six half-midbrain

samples was frozen in one of the following combinations

of temperature: (I) placement at -20 C for 30 min, fol-

lowed by -80 C storage; (II) placement and storage at

-80 C; and (III) snap freezing in liquid nitrogen, followed

by -80 C storage (Table 1), resulting in 2 samples of

each frozen method. One slice of 10 lm was obtained from

each sample and stained with cresyl violet. Three inde-

pendent investigators blinded to the congelation method

Fig. 1 Principles of laser microdissection. The isolation and collec-

tion of specific cells is based on a laser. First, a laser microdissection

is performed. Second, a laser pulse catapults the selected sample into

a collection device. Using this technique, it is possible to achieve

contact-free isolation of specific areas, single cells and even specific

cell compartments

994 M. Molina et al.

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qualitatively analyzed the slices based on the morphology

of neurons and quality of the tissue after sectioning and

staining optimization.

Sectioning and staining the tissue for laser

microdissection

Two frozen midbrains were sectioned in sagittal plane atthe level of the substantia nigra. Sectioning was performed

with a Cryostat Microm HM550 (Thermo Scientific,

Dreieich, Germany) with a fixed knife holder (Leica

Biosystems, Nußloch GmbH, Nußloch, Germany) at

-10 C object temperature and -20 C chamber temper-

ature. Before sectioning, tissue was left in the cryostat for

15 min for warming to-20 C. The sections (5, 10, 20 and

30 lm) were placed on special membrane slides for LMD

(1.0 PEN membrane slides, Carl Zeiss Microscopy GmbH,

Göttingen, Germany). To distinguish the cells, sections

were stained with cresyl violet according to a protocol from

Carl Zeiss Microscopy GmbH (Carl Zeiss MicroImaging,LCM Protocols—Protein Handling for LC/MS), with slight

modifications. Cresyl violet stains the nuclei deep violet

and the cytoplasm weak purple. This staining technique is

recommended by Zeiss for laser microdissection intended

for subsequent proteomic approaches. Briefly, 1 g of cresyl

violet (cresyl violet acetate, Sigma Life Sciences, St. Louis,

USA) was diluted in 100 ml of 50 % ethanol, with stirring

overnight on a stirring plate. The following day, the solu-

tion was filtered to remove undissolved particles. All

solutions were freshly prepared and used precooled (4 C).

A summary of the staining protocol is provided in Table 2.

Laser microdissection

In the LMD machine, a laser pulse transports the selected

sample out of the slide into a collection device (usually a

microtube cap, which may be filled with a solution). With

this approach, we tested two different solutions for wet

collection: ultrapure water (TKA-Gene PURE, Thermo

Scientific, Baltimore, USA) and 50 % acetonitrile (Bio-

solve BV, Valkenswaard, Netherlands) diluted in water.

LMD was performed with a PALM Micro Beam

instrument (P.A.L.M.-System LCM, Carl Zeiss Micro-

scopy GmbH) with non-adhesive tubes (MicroTube 500,

Carl Zeiss Microscopy GmbH). The microtube cap was

filled with 50 ll of one of the solutions. After finishing

LMD, the tube was handled, closed and stored upside-

down at -80 C. Because we intended to perform mass

spectrometry, 2.5 ll of 2 % RapidGestTM SF Surfactant

(Waters GmbH, Milford, MA, USA) was carefully addedbefore storage for a final concentration of 0.1 %.

The LMD was tested on 5, 10, 20 and 30 lm thick

sections to collect single cells as well as whole brain

regions of the tissue sections.

Before placing a sample cap over the PEN membrane

slide, the regions/cells to be collected were marked; if all of

the desired samples could not be collected from one slide,

the next slide was also used to prevent unnecessary evap-

oration of the solution in the tube cap. The selection of

optimal thickness was based on the ability to catapult the

region/cells, preventing falling apart or attachment to the

membrane, and the ability to establish the laser parametersthat would enable an effective catapult process within a

short time.

Sample preparation and sample digestion for mass

spectrometric analysis

For sample digestion, the tubes containing the collected

tissue were incubated upside-down in an ultrasonic bath for

1 min, and then, the samples were centrifuged briefly. This

step was repeated. Next, the samples were incubated at

95 C for 5 min in a thermomixer and centrifuged again.

One microliter of 250 mM 1,4-dithiothreitol (DTT,

AppliChem GmbH, Darmstadt, Germany) was added to

each sample, and the mixture was incubated for 30 min at

60 C. The samples were then incubated with 1.4 ll of

0.55 M iodoacetamide (AppliChem) at room temperature

for 30 min in the dark. Digestion was initiated by addition

of 1:4 trypsin in water (Promega, Mannheim, Germany) at

37 C for 4 h and stopped with 3.25 ll of 10 % TFA for

30 min at 37 C. The samples were then centrifuged for

15 min at 14,000 rpm and 4 C. The resulting supernatant

was transferred into a new sample cap and dried with

SpeedDry (RVC 2-25 CDplus, Martin Christ

Gefriertrocknungsanlagen GmbH, Osterode, Germany).

Finally, 30 ll of 0.1 % TFA was added to each sample.

Protein concentration determination with amino

acid analysis

After sample digestion, protein concentrations were

determined by amino acid analysis as described by Plum

et al. (Plum et al. 2013). For this process, 5 ll of each

digested sample was dried completely in a glass tube. Next,

Table 1 Overview of the freezing procedures

Case Side Procedure

1 Left -20 C/ -80 C

Right -80 C

2 Left -20 C/ -80 C

Right Liquid nitrogen/ -80 C

3 Left -80 C

Right Liquid nitrogen/ -80 C

Enrichment of single neurons and defined brain regions from human brain tissue samples for … 995

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the samples were dissolved by the addition of 10 ll of

20 mM HCl. According to the manufacturer’s instructions,

an amino acid analysis was performed using the Acquity

HPLC and AccQ-Tag Ultra system (Waters GmbH). For

derivatization and to allow the conversion of primary and

secondary amines into stable derivatives, the samples were

incubated with 10 ll of AccQ-Tag reagent and 30 ll of

internal norvaline standard (final concentration: 10 pmol/

ll) for 10 min. Amino acid derivatives were separated on

an AccQ-Tag Ultra RP column and detected by an Acquity

UPLC-TUV detector (Waters GmbH). The amino acids

were quantified using 10 pmol/ ll amino acid standards.

Mass spectrometric analysis

The LC–MS/MS analysis was performed on an UltiMate

3000 RSLC nano LC system (Dionex, Idstein, Germany)

coupled to the Q Exactive system (Thermo Fisher Scien-

tific, Bremen, Germany). Sample loading on the trap col-

umn (100 lm 9 2 cm, particle size 5 lm, pore size 100 Å,

C18, Thermo Scientific) was performed by an autosampler

at a flow rate of 30 ll/min at 60 C with 0.1 % TFA. After

a washing step, the sample was loaded onto an analytical

C18 column (75 lm 9 50 cm, particle size 2 lm, pore

size 100 Å, Thermo Scientific). For peptide separation, a

linear gradient of 4–40 % running buffer B (84 % ACN,

0.1 % FA; running buffer A: 0.1 % FA) was conducted for

95 min, followed by a washing step at 95 % B for 5 min

and an equilibration step from 95 to 4 % B. The connection

between the HPLC system and Q Exactive was online, and

electrospray ionization was performed. The ion spray

voltage was set to 1600 V (?) and the capillary temperature

to 250 C. The scan range was defined as 350–1400 m/z

for the full scan mode, and for SIM scans, an Orbitrap res-

olution of 70,000 (at 200 m/z) was set. Internal recalibration

useda target AGC of3e6,fill timeof 80 msand the lock mass

of polydimethylcyclosiloxane (m/z 445.120). The settings

for the initiation of MS/MS analysis of m/z values were as

follows: dynamic exclusion list: 30 s, top 10 ions (charge,

?2, ?3, ?4).

Fragments for MS/MS analysis were generated using

high-energy collision-induced dissociation (HCD). For ion

dissociation, a normalized collision energy (NCE) of 27 %,

isolation window of 2.2 m/z and a fixed first mass of

130 m/z were used. In addition, the following fragment

analysis was performed in an Orbitrap analyzer with a

resolution of 35,000 at 200 m/z, target of 1e6 and fill time

of 120 ms.

The resulting RAW data of the LC–MS/MS analysis

was interpreted by Proteome Discoverer (version

1.4.0.288) using the UniProt database [UniProt/SwissProt-

Release 2013_05 of 01.05.2013; 541,561 (http://www.uni

prot.org)] as a protein sequence reference. The taxonomy

was set to Homo sapiens and a precursor mass tolerance of

5 ppm and fragment mass tolerance of 20 amu were

applied. The target FDR was set to 0.01. Additionally, the

following dynamic modifications were assumed: oxidation

and carbamidomethylation. The resulting MGF files were

analyzed with Protein Inference Algorithms software (PIA;

version 10.0-rc2-dev, http://www.ruhr-uni-bochum.de/

mpc/software/PIA/index.html, https://github.com/mpc-

bioinformatics/pia) and Ingenuity Pathway Analysis

software (IPA; version 18488943) (http://www.ingenuity.

com/products/ipa) using the core analysis tool.

Results

In this paper, we describe an approach for studying single

human neuron populations using LMD technology. This

approach is effective for proteomic application. Although

we focused on the substantia nigra of control human

brains, this approach is also suitable for other cell regions

and single cell types in humans and animal models.

Definition of the optimal anatomical plane

for neuron collection within the substantia nigra

Sagittal planes were selected to allow recognition of the

layers of the substantia nigra pars compacta and neurons.

Furthermore, the sagittal plane is the most appropriate

plane of section because the long axis of a majority of

nigral neuronal perikarya is arranged in the rostro-caudal

plane (Fig. 2).

Table 2 Cresyl violet staining

procedure Step Solution Procedure Duration Temperature

1. 70 % ethanol Incubation 2 min 4 C

2. 1 % cresyl violet Incubation 30 s RT

3. – Discard remaining solution

4. 70 % ethanol Dipping 3–5 9 1 s 4 C

5. 100 % ethanol Dipping 1 9 s 4 C

6. – Air dry 1–2 min RT


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http://www.uniprot.org/http://www.uniprot.org/http://www.ruhr-uni-bochum.de/mpc/software/PIA/index.htmlhttp://www.ruhr-uni-bochum.de/mpc/software/PIA/index.htmlhttps://github.com/mpc-bioinformatics/piahttps://github.com/mpc-bioinformatics/piahttp://www.ingenuity.com/products/ipahttp://www.ingenuity.com/products/ipahttp://www.ingenuity.com/products/ipahttp://www.ingenuity.com/products/ipahttps://github.com/mpc-bioinformatics/piahttps://github.com/mpc-bioinformatics/piahttp://www.ruhr-uni-bochum.de/mpc/software/PIA/index.htmlhttp://www.ruhr-uni-bochum.de/mpc/software/PIA/index.htmlhttp://www.uniprot.org/http://www.uniprot.org/


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Freezing protocol for tissues used for LMD

processing

We tested multiple freezing protocols to preserve tissue

morphology and integrity. A comparison of the protocols is

shown in Fig. 3. Direct storage of the tissue at-80 C and

preliminary freezing at -20 C before storage at -80 C

lead to tissue disruption, which adversely affected tissuemorphology and integrity. In contrast, a quick submersion

in liquid nitrogen prior to storage at -80 C resulted in a

more integrated tissue sample. Therefore, this protocol was

used.

Testing of section thickness for neuron analysis

using LMD

Tissue section thickness is a crucial factor in achievinghigh quality and repeatable samples when using LMD for

400705948_93_40_2.5_SubnigCompIntermedVentralTierventral

pars compacta

pars diffusa

Fig. 2 Optimal plane for

isolation of neurons from

substantia nigra tissue sections.

Sagittal sections of substantia

nigra tissue are optimal for the

isolation of neurons because the

different tiers of the substantia

nigra pars compacta are easily

identified. The inset shows

neurons in the substantia nigra

pars diffusa from a 400 lmthick gallocyanin-stained

section (Heinsen et al. 2000)

Fig. 3 Optimal freezing procedure for laser microdissection. The

freezing process for LMD processing was optimized. a The resulting

tissue after freezing the sample at -20 C before storage at -80 C is

shown. b Visualizes the tissue after it was stored directly at -80 C.

c The results of tissue preparation using a quick incubation with liquid

nitrogen before the tissues were stored at -80 C are shown. Tissue

morphology and integrity was highly affected when the tissue was

stored directly at -80 C (b) and when the tissue was stored

preliminary at -20 C before it was stored at -80 C (a). The best

storage results and intact tissue were obtained when the tissue was

immersed for a few seconds in liquid nitrogen before storage at

-80 C (c)


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sample collection. Therefore, we tested four different

thicknesses (5, 10, 20 and 30 lm). Using the ‘‘Cutting’’

and ‘‘RoboLPC’’ settings of the LMD, we compared the

results of each tissue thickness. Figure 4 shows that the

accuracy of laser cutting decreased with increasing thick-ness. Based on these results, we used 10–20 lm sections

for selected regions and 5–10 lm sections for single cell

isolations because a higher accuracy was needed.

Staining procedure for LMD

Cresyl violet is a common stain for neurons (Mouledous

et al. 2002). Therefore, this method was tested for LMD

application. This staining provided easy and fast handling

of the samples, and it did not show any complications in

background quality or optical resolution. These results

supported our use of cresyl violet as the staining choice forLMD processing.

Adjustments for laser microdissection

LMD is a process that depends on several parameters, each

of which must be optimized for each tissue type and

sample. The settings are influenced by the tissue type, the

cells or regions to be isolated, the thickness of the tissue

and the applied magnitude. Therefore, it is essential to

investigate these settings for each sample collection. Wang

et al. (2009) described that a higher magnitude or thicker

section requires more energy for cutting.

Isolation of cells using LMD

Single cells from the 5 and 10 lm sections were dissected,

without difficulty, using a thin cutting line (indicating the

best laser energy setting), and laser energy adjustments

were achieved quickly. The catapult method was efficient

for all attempts.

Isolation of regions using LMD

Energy setting adjustments took longer and resulted in

broader cutting lines for LMD of single cells from 20 lm

sections, and some of the catapult sections were not suc-cessful. However, 20 lm sections enabled effective and

specific capture of isolated entire brain regions, such as the

substantia nigra.

Area selection for mass spectrometry analysis

The area that must be isolated for a proper mass spectro-

metric analysis must be investigated. The required area

depends on the tissue and cell type and tissue thickness. At

Fig. 4 Optimal section thickness for cutting single cells and regions.

Section thickness is essential for optimal neuron isolation using laser

microdissection. Therefore, four different thicknesses were tested:

5 lm (a), 10 lm (b), 20 lm (c) and 30 lm (d). For each thickness, a

cutting line and square were cut. Additionally, a square was

catapulted after cutting to ensure that accurate laser microdissection

processing was possible. An increase of section thickness decreased

cutting accuracy. Using 5 lm sections, a thin fine cutting line was

possible and catapulting of the sample occurred without difficulties.

However, the cutting line broadened and a dark edge appeared with

increasing section thickness (20 lm). Neither cutting nor catapulting

of the sample was possible in 30 lm sections. Testing revealed that

cutting conditions were excellent in 5 and 10 lm sections


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least 100 ng protein amount must be employed for a good

mass spectrometric analysis. Therefore, a region of

25,000,000 lm2 was sufficient to analyze the entire sub-

stantia nigra, in sections that had a thickness of 20 lm, by

mass spectrometry. A total of 2500 neurons

(*1,500,000 lm2) were isolated in 10 lm thick sectionsfor mass spectrometric analysis of single neurons.

Isolation of tissue sample using LMD

Cells for proteomic analysis must be isolated in an opti-

mized solution. We tested two different solutions (water

and 50 % acetonitrile) using the entire substantia nigra.

Mass spectrometric analysis revealed equal amounts of

identified proteins after isolation in water as well as 50 %

acetonitrile. We selected water as the collecting buffer

because no relevant differences in the amount of identified

protein groups were observed.

Sample preparation protocol for mass spectrometry

The presented protocol performed a standard digestion

with trypsin to investigate the substantia nigra and a

number of identified proteins, as well as analyze their

locations and types within this tissue. We performed a

successful mass spectrometric analysis using this protocol

and identified 1144 proteins within the substantia nigra

in 500,000,000 lm3 tissue. The left part of Fig. 5a, c

summarizes the results of this analysis, which was per-

formed to provide an initial overview of the substantia

P r o t e i n

t y p e

L o c a l i z a t i o

n

Tissue Neurons

a b

c d

Fig. 5 Summary of mass spectrometry results. Substantia nigra

tissue isolated by laser microdissection was analyzed successfullyusing mass spectrometry, which revealed the first overview of the

substantia nigra proteome. A total of 1144 proteins were identified

(out of 500,000,000 lm3) in the entire substantia nigra tissue, which

were investigated according to their location (a) and type (c). Both

factors are of special interest to reveal membrane-specific proteins

that greatly impact cell homeostasis and brain function. We revealed

that 15 % of all identified proteins were located on the plasma

membrane and that 13 % of proteins were associated with transporter(11 %) or channel proteins (2 %). Mass spectrometric analysis of

15,000,000 lm3 isolated neurons located in the substantia nigra

resulted in the same distributions (b). Proteins that were associated

with transporter (12 %) and channel proteins (1 %) composed 13 %

of the total amount of identified protein groups (d)


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nigra proteome. The greatest quantities of proteins were

located in the cytoplasm (66 %) (Fig. 5a). Proteins that

were associated with the plasma membrane made up

15 % of the total identified protein groups. We also

analyzed the distribution of protein types (Fig. 5c). In

total, 13 % of identified proteins were successfully shown

to be associated with transporter (11 %) and channel

proteins (2 %).Mass spectrometric analysis of isolated neurons located

in the substantia nigra (15,000,000 lm3) resulted in the

same distributions (right part of Fig. 5b, c) of 303 identi-

fied proteins. A total of 71 % of proteins were located in

the cytoplasm. Fourteen percent of proteins were associ-

ated with the plasma membrane (Fig. 5b). Proteins that

were associated with transporter (12 %) and channel pro-

teins (1 %) comprises 13 % of the total identified proteins

(Fig. 5d). These results indicate that our digestion protocol

is suitable for proteomic brain investigations.

Discussion

A combined technique of single cell dissection and sub-

sequent molecular analysis could facilitate better under-

standing of the disease mechanisms of neurodegenerative

diseases such as Alzheimer’s and Parkinson’s disease.

Common features of many neurodegenerative diseases are

protein aggregation and the selective degeneration of a

particular group of neurons (Dickson 2007; Duyckaerts

et al. 2009). Therefore, the molecular analysis of single

neuronal cell populations is expected to yield a better

understanding of disease mechanisms. Although several

groups in the past few years have used proteomics and

other molecular biology techniques to study the brain and

the mechanisms underlying neurodegenerative diseases,

many questions still remain unanswered (Dumont et al.

2006; Kitsou et al. 2008; Werner et al. 2008; He et al.

2006). Changes in specific neuronal populations are com-

monly masked in global analyses, where whole brains or

large sections rather than specific cell groups are homog-

enized for analysis.

LMD was developed less than 20 years ago and enables

the isolation of specific cells within a tissue (Emmert-Buck

et al. 1996). In combination with new analytical methods

allowing for the analysis of small sample volumes, new

insights into the pathological cascade, therapy options, and

neuroprotective agents, preclinical/clinical biomarkers are

possible. However, the combination of these recent tech-

niques can have various limitations depending on the aims

of the study.

In the present study, by focusing on the substantia nigra

of elderly control human brains, we aimed to overcome

several technical hurdles described below.

Optimized freezing process for preserving tissue

morphology and tissue integrity

The use of fresh tissue is mandatory for proteomics with

LMD. Fresh tissue has no changes in RNA (Goldsworthy

et al. 1999) or proteins (Rekhter and Chen 2001) compared

with formalin-fixed paraffin-embedded tissue, which con-

tains protein cross-linking products. Additionally, paraffinis not compatible with LC–MS/MS and requires a

deparaffination step, resulting in protein loss (Hood et al.

2006). Therefore, a freezing procedure for LMD workflows

had to be established. We tested multiple freezing proto-

cols, and the best results were achieved when the sample

was incubated for a short time in liquid nitrogen before

storage at -80 C. Slow freezing promotes ice crystal

formation of water within the tissue, which expands and

disrupts the tissue. Therefore, it is important to freeze the

tissue rapidly to prevent water crystallization. The use of

liquid nitrogen allows water to transform into a vitreous

form that does not expand.

Optimal plane for analyzing neurons

within the substantia nigra

Because there is a lack of validated normative data from

morphological studies of the substantia nigra, especially for

older individuals, we utilized a tissue-processing method

based on celloidin mounting to establish an optimal plane for

collecting neurons (Cabello et al. 2002). Cytoarchitectoni-

cally, thesubstantia nigra is dividedinto three parts: the pars

compacta, pars diffusa, and pars reticulata (Braak andBraak 1986). The neurons containing neuromelanin are in the pars

compacta, which consists of layers of medium to large

neurons (Braak and Braak 1986). To improve sampling

strategy, theneuroanatomyof the region wasconsidered, and

a sagittal plane was selected to allow recognition of the

layers and neurons for single cell analysis. Further, the

sagittal plane is the most appropriate plane of section

because the long axis of a majority of nigral neuronal peri-

karya is arranged in the rostro-caudal plane (Fig. 2).

Optimal section thickness for neuron analysis using

LMD

Sectioning is a crucial step for LMD-based studies. In

particular, section thickness is essential for good and

repeatable LMD processing. Testing of four different

thicknesses (5, 10, 20 and 30 lm) revealed that the accu-

racy of laser cutting decreases with increasing thickness

(Fig. 4). These results agree with those of Wang and

coworkers. These authors needed higher energies for 20 lm

sections than for 10 lm sections, and to make multiple cuts


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to sever the tissue. However, they used this cutting proce-

dure for cell groups rather than single cells (Wang et al.

2009), indicating that the cutting accuracy for 5 and 10 lm

sections is excellent and that, even very small single cells

can be isolated without damaging the tissue (Fig. 4).

Optimal staining procedure

Staining is a necessary technique for distinguishing dif-

ferent tissue patterns and cell types, and is an important

part of LMD experiments. To obtain a better signal and

minimize background, the sample can be incubated with a

higher concentration of staining solution; however, this

procedure may negatively affect the proteomic analysis

(Gutstein and Morris 2007). The literature contains

numerous staining protocols for different stains in the

context of LMD processing. Common stains include

hematoxylin and eosin (De Souza et al. 2004; Dos Santos

et al. 2007), toluidine blue (Sridharan and Shankar 2012;

Kulkarni et al. 2013; Kirana et al. 2009; Lawrie et al. 2001;Mouledous et al. 2002) and cresyl violet (Boone et al.

2013; Aaltonen et al. 2011). Immunohistochemical staining

is also popular for LMD processing because it can distin-

guish single cell types (e.g. astrocytes from small neurons)

or cellular subtypes (e.g. dopaminergic from GABAergic

neurons). The choice of staining procedure depends not

only on sample tissue but also on the additional sample-

processing procedures and the study aims. Previous

investigations have shown that hematoxylin and eosin

staining is not compatible with proteomic analysis. For

instance, Gutstein and coworkers tested different conven-

tional stainings on brain tissue, and showed that hema-

toxylin and eosin is not compatible with 2D gel analysis

(Gutstein and Morris 2007). Several other groups have

confirmed the negative influence of hematoxylin/eosin

staining on the proteome (Sitek et al. 2005; Craven et al.

2002; Craven and Banks 2001). However, this stain has

shown no effects on samples for RNA and DNA investi-

gations (Burgemeister et al. 2003). Toluidine blue is often

used for the DNA and RNA analysis of LMD samples

(Kulkarni et al. 2013) and can also be used for proteomic

analysis (Lawrie et al. 2001; Mouledous et al. 2002). In

contrast, Craven and coworkers investigated kidney sam-

ples with toluidine blue and identified detrimental effects

on protein recovery in 2D gel analysis (Craven et al. 2002).

Cresyl violet is especially common for staining neurons. It

is a cationic solution (Mouledous et al. 2002) and binds to

acidic components of the neuronal cytoplasm, especially

ribosomes, which are present in large numbers in neurons

(Burnet et al. 2004; Eltoum et al. 2002). Clément-Ziza

et al. (2008) improved the cresyl violet staining procedure

for RNA analysis by replacing all solvents with ethanol,

which prevents sample degradation. Compared with other

common stains, cresyl violet provides the best contrast

between stained and unstained tissue (Ginsberg and Che

2004). The staining of frozen tissue did not show any

complications. The tissue background was transparent and

resulted in an excellent optical resolution. Based on the

literature described above all further experiments were

performed on cresyl violet-stained samples.

Optimized laser microdissection

Further, LMD performance is influenced by temperature

and humidity, which due to weather variability changes the

energy settings daily. Therefore, it is especially important

to optimize the sample amplitude and thickness for each

sample. The use of a higher magnitude and thinner section

is advisable for single cells or small areas to ensure that no

surrounding tissue is collected.

Furthermore, the tissue amounts required to achieve the

amount of proteins needed for mass spectrometric analysis

strongly depends on the tissue type. Therefore, it isessential to investigate these parameters during pre-trials.

Optimized sample preparation protocol for mass

spectrometry

Sufficient tissue of at least 100 ng must be collected to

guarantee a mass spectrometric analysis of high perfor-

mance. This study achieved proper mass spectrometric

analysis using 500,000,000 lm3 of substantia nigra tissue

and 15,000,000 lm3 of isolated neurons located in the

substantia nigra. However, the required area depends on

the tissue and cell type, and it must be investigated for each

tissue type individually. Therefore, preliminary tests are

essential at the start of a study. For example, to define the

amount of neurons that are needed for mass spectrometric

analysis, 5000 neurons were set as starting point for

investigations, revealing that a total of 2500 neurons

(*1,500,000 lm2) in 10 lm thick sections are sufficient

for mass spectrometric analysis of single neurons. How-

ever, the numbers of identified proteins for isolated neurons

could be enhanced if more neurons are isolated.

Further, it is very important to establish a specific and

reproducible sample preparation protocol to achieve com-

parative proteome analyses. In this context, the digestion

step is crucial. Tryptic digestion is commonly used in

proteomic research, and this technique produced good

results for LMD processing. Using this protocol, proteins

could be identified that are associated with the substantia

nigra as well as Parkinson’s disease (Spillantini et al. 1997;

Bonifati et al. 2003; Damier et al. 1999). For example, a-

synuclein was identified with sequence coverage of

46.43 %. Nine unique peptides of DJ-1 were identified, a

protein that is associated with familial PD, resulting in


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sequence coverage of 58.20 %. Further, a dopamine

transporter {uniprot ID Q01959 [UniProt/SwissProt-Re-

lease 2013_05 of 01.05.2013; 541,561 (http://www.uniprot.

org)]} was also detected.

Tryptic digestion should be strongly considered in

studies concerning membrane associated proteins. Mem-

brane proteins, which are present in the plasma membrane

and subcellular compartments, strongly impact signalingprocesses. Therefore, they are highly relevant for cell

homeostasis and brain function, and are highly relevant in

proteomic research, especially of transporter, receptor and

channel proteins. However, different digestion enzymes or

chemical cleavage should be tested depending on the

interest of the study. For example, cyanogen bromide is a

commonly used chemical substance. Helling et al. (2012)

used this type of sample preparation for phosphoproteome

analysis of the cytochrome c oxidase membrane protein

complex and improved the mass spectrometric analysis of

integral membrane proteins. The use of cyanogen bromide

identified six new phosphorylation sites that were not foundwhen the samples were digested with trypsin. Chy-

motrypsin is another possible chemical for digestion.

(Fischer et al. 2006) tested different digestion protocols to

investigate the membrane proteome of Corynebacterium

glutamicum. A digestion mixture containing trypsin and

chymotrypsin increased the identification of proteins that

contained large hydrophobic domains. In addition, predi-

gestion of the sample with trypsin decreased the amount of

soluble proteins that could mask low abundant membrane

proteins (Fischer et al. 2006). Nevertheless, solvents may

also impact digestion and optimization may be a key factor

to improve the digestion protocol (Russell et al. 2001).

Pitfalls for LMD-based proteomics

Although LMD is a promising tool for proteomic studies of

brain tissues and especially of different neuronal cell types,

there are some pitfalls that have to be considered con-

cerning LMD processing. First, to obtain an adequate mass

spectrometric analysis, frozen-fresh tissue must be used.

The use of formalin-fixed paraffin-embedded tissue can

negatively influence the results. During the formalin fixa-

tion process, proteins undergo degradation and cross-link-

ing (Azizadeh et al. 2015). These modifications can hinder

accurate mass spectrometric analysis.

Concerning frozen-fresh tissue, it has to be ensured that

the samples are always stored on ice to prevent protein

degradation or modifications. Cooling of samples during

LMD process is not possible. Therefore, only one section

of tissue should be processed at a time.

To prevent disturbances during mass spectrometry, a

sample cap should be used that can be filled with a col-

lecting buffer instead of silicon filled caps.

In addition, the tissue sample adhered to the slide must

be completely dry to guarantee catapulting of the sample.

The cutting line of the laser must be thin and accurate to

avoid contamination, and the catapulting energy must be

high enough to ensure that the tissue sample reaches the

sample cap.

Outlook

Compared with traditional cell-isolation strategies, LMD

offers outstanding possibilities for understanding brain

function under normal and diseased conditions, as well as

during the aging process. Changes in specific neuronal

populations, which are normally masked in global

Sampling

Freezing and Storage

(liquid nitrogen -80°C)

Cryostate secons (in sagial orientaon)

20 µm ckness (whole ssue analysis )

5-10 µm ckness (single cells analysis)

Staining

(Cresyl violet)

Laser microdissecon

Sample preparaon for mass spectrometric

analysis

(trypc digest)

Mass spectrometric analysis

Fig. 6 Summary of the laser microdissection workflow. This figure

demonstrates the resulting laser microdissection workflow of our

study for human substantia nigra tissue


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analyses, can be analyzed in a highly specific manner,

enabling insights into questions such as why specific

neurons are affected in Parkinson’s disease or why the

clinical symptoms of Alzheimer’s disease can greatly

vary among people with similar neuropathological char-

acteristics. The answers to these questions are integral in

developing clinical biomarkers, therapeutic interventions,

and potential neuroprotective agents. LMD, in combina-tion with modern analysis techniques such as mass spec-

trometry may address some of these questions. Further,

the possibility to combine LMD with other fractionation

strategies could reveal a deeper insight in molecular

processes. To continue with the example of substantia

nigra analysis, a purification and enrichment of neu-

romelanin granula or mitochondria out of laser

microdissected neurons (Plum et al. 2014) could give a

better understanding of molecular processes in cells.

LMD has many advantages and is simple to execute. We

demonstrated that this approach (summarized in Fig. 6) in

combination with modern analysis techniques can be usedto characterize the neuronal proteome, and help to reveal

the pathophysiological mechanisms within healthy and

neurodegenerative affected brains.

Acknowledgments This work was supported by WTZ Brasilien, a

project of the BMBF, Germany (01DN14023), Conselho Nacional de

Desenvolvimento Cientı́fico e Tecnológico (CNPQ), Fundação de

Amparo à Pesquisa do Estado de São Paulo (Fapesp), P.U.R.E.

(Protein Unit for Research in Europe), a project of Nordrhein-West-

falen, a federal German state, and the HUPO Brain Proteome Project.

The authors gratefully thank the Brazilian Brain Bank in São Paulo

for providing tissues and Ulrich Sauer, Volker Wollscheid, Lukas

Baran, Ulrike Weber and Gabrielle Friedemann from Carl Zeiss

Microscopy GmbH, Munique, Germany, for technical support. Fur-

thermore, we would like to thank Pascal C. Rauher for providing

Fig. 1.

Conflict of interest There are no conflicts of interest to report.

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