Journal of Neuroscience Methods 263 (2016) 16

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Journal of Neuroscience Methods

journal homepage: www.elsevier.com/locate/jneumeth

Basic neuroscience

Isolation of functionally active and highly puried neuronal

mitochondria from human cortex
Nicolas K. Khattar a , Svitlana Yablonska a , Sergei V. Baranov a , Oxana V. Baranova a ,
Eric S. Kretz a , Timothy M. Larkin a , Diane L. Carlisle a , R. Mark Richardson b,c ,
Robert M. Friedlander a,c,
a

Neuroapoptosis and Translational Therapeutics Laboratory, Department of Neurological Surgery, University of Pittsburgh, Pittsburgh, PA, United States
Brain Modulation Laboratory, Department of Neurological Surgery, University of Pittsburgh, Pittsburgh, PA, United States
c
University of Pittsburgh Medical Center, Pittsburgh, PA, United States
b

h i g h l i g h t s
Combined Percoll gradient centrifugation and anti-TOM22 magnetic bead extraction is used to isolate mitochondria.
The mitochondria obtained are neuronal (from synaptosome disruption).
The mitochondria obtained have minimal cytoplasmic contaminants (plasma membrane, peroxisomes, lysosomes, synaptosomes, endoplasmic
reticulum).

The mitochondria are functionally active based on measurements of respiration and protein import.

a r t i c l e

i n f o

Article history:
Received 28 September 2015
Received in revised form 9 January 2016
Accepted 14 January 2016
Available online 22 January 2016
Keywords:
Neurons
Mitochondria
Human cortex
Isolation
Percoll
Synaptosome
Non-synaptosomal mitochondria
Differential centrifugation
Puried
Functional

a b s t r a c t
Background: Functional and structural properties of mitochondria are highly tissue and cell dependent,
but isolation of highly puried human neuronal mitochondria is not currently available.
New method: We developed and validated a procedure to isolate puried neuronal mitochondria from
brain tissue. The method combines Percoll gradient centrifugation to obtain synaptosomal fraction
with nitrogen cavitation mediated synaptosome disruption and extraction of mitochondria using anti
mitochondrial outer membrane protein antibodies conjugated to magnetic beads. The nal products of
isolation are non-synaptosomal mitochondria, which are a mixture of mitochondria isolated from different brain cells (i.e. neurons, astrocytes, oligodendrocytes, microglia) and synaptic mitochondria, which
are of neuronal origin. This method is well suited for preparing functional mitochondria from human
cortex tissue that is surgically extracted.
Results: The procedure produces mitochondria with minimal cytoplasmic contaminations that are functionally active based on measurements of mitochondrial respiration as well as mitochondrial protein
import. The procedure requires approximately four hours for the isolation of human neuronal mitochondria and can also be used to isolate mitochondria from mouse/rat/monkey brains.
Comparison with existing methods and conclusions: This method will allow researchers to study highly
enriched neuronal mitochondria without the confounding effect of cellular and organelle contaminants.
2016 Elsevier B.V. All rights reserved.

1. Introduction

Corresponding author at: Department of Neurological Surgery, University of

Pittsburgh School of Medicine, UPMC Presbyterian Hospital, Fourth Floor, Suite B449,
Pittsburgh, PA 15213, United States. Tel.: +1 412 647 6358; fax: +1 412 864 3284.
E-mail address: friedlanderr@upmc.edu (R.M. Friedlander).
http://dx.doi.org/10.1016/j.jneumeth.2016.01.017
0165-0270/ 2016 Elsevier B.V. All rights reserved.

Mitochondria play an important role in neuronal homeostasis by providing energy and participating in numerous signaling
pathways (Zhang et al., 2013; Yano et al., 2014; Friedlander, 2003;
Hensley and Harris-White, 2015; Wang et al., 2008). Mitochondrial function has been shown to be different in various tissues and
cell types (Carafoli and Lehninger, 1971; Zhang et al., 2013; Teng
et al., 2004; Zhou et al., 2014). Given that mitochondrial dysfunction

N.K. Khattar et al. / Journal of Neuroscience Methods 263 (2016) 16

plays a critical role in neurodegeneration, it is of outmost importance to accurately evaluate highly puried functional neuronal
mitochondria (Zhu et al., 2002; Zhang et al., 2008; Kristal et al.,
2004). Isolation of highly puried functional neuronal mitochondria has been very challenging due to the heterogeneity of brain
tissue. The only method shown to be able to provide neuronal mitochondria involves their extraction from synaptosomes.
Synaptosomes are vesicles formed from neuronal processes during mechanical homogenization of brain tissue and encapsulate
neuronal mitochondria. It has been shown that synaptosomal
mitochondria respond differently to stressors such as Ca2+ overload as compared with non-synaptosomal mitochondria (Brown
et al., 2006). Our group has recently reported that mitochondrial
protein import is impaired exclusively in synaptosomal (or neuronal) mitochondria of presymptomatic R6/2 mice, a mouse model
of Huntingtons disease (HD) (Yano et al., 2014). It is of interest
that primary neurons and cell lines expressing mutant huntingtin
demonstrated a mitochondrial protein import defect as well (Yano
et al., 2014).
The most robust protocols to isolate synaptosomal mitochondria include a Percoll/Ficoll discontinuous gradient centrifugation
step followed by synaptosomal disruption with either digitonin
or nitrogen cavitation pressurization (Sims and Anderson, 2008;
Kristian, 2010; Hansson et al., 2008; Barksdale et al., 2010). Neither method, however, allows for the elimination of contaminants,
including synaptic vesicles, lysosomes, and peroxisomes. Whole
synaptosomes are also still found in the nal mitochondrial fraction
using these methods (Kristian, 2010). To mitigate this obstacle, we used specic anti-mitochondrial outer membrane protein
TOM22 antibodies to purify synaptosomal mitochondria without
loss of function. These antibodies are conjugated with magnetic
microbeads for use in magnetic activated cell sorting (MACS). Using
a magnetic eld, the microbeads are able to selectively retain
mitochondria as they bind to the TOM22 antibodies. Any nonmitochondrial fraction of the sample will therefore be washed out.
The exclusive use of the MACS technique was shown to be sufcient to obtain brain mitochondria from a mixture of cell types
(neuronal body, glia) but not pure neuronal mitochondria because
it requires disruption of the synaptosomes before application of
the antibodies. The method described in this report yields highly
puried and functional neuronal mitochondria. We successfully
characterized the mitochondrial status of human cortical neurons
using mitochondria obtained from this protocol. Previous reports
described brain mitochondria isolation (Hansson et al., 2011). To
our knowledge, this is the rst reported protocol to isolate highly
pure functional neuronal mitochondria from adult human cortex.
This report describes the isolation of pure synaptosomal/neuronal
mitochondria. It also yields highly puried non-synaptosomal
mitochondria that can be used for functional or structural
analyses.

2. Materials

2.2. Equipment
Motor-driven Teon Potter Elvehjem homogenizer (Sigma, cat. no.
Z403903)
50 mL round bottom transparent centrifuge tubes (Nalgene, cat.
no. 3138-0050)
16 mL polycarbonate round bottom centrifuge tubes for the F21850y rotor (18 mm 100.6 mm; Nalgene, cat. no. 3117-0160)
1.7 mL microcentrifuge tubes (Avant, cat. no. 2925)
Refrigerated hi-speed centrifuge with xed-angle rotor (Thermo,
Sorvall RC-6, F21-850y rotor or any compatible hi-speed centrifuge).
Cell disruption vessel (Parr Instrument Company, cat. no. 4639).
Mitochondria Isolation Kit for human tissue, which includes antiTOM22 microbeads, 10 separation buffer, MACS separation LS
Columns, and magnetic Quadro MACS Separator (Miltenyi Biotec,
cat. no. 130-094-532).
Nitrogen tank
2.3. Reagent preparation
Isolation Buffer 1 (IB-1): 225 mM sucrose, 75 mM mannitol, 1 mM
EGTA, 5 mM HEPES adjusted to pH 7.4 with Tris base.
Isolation Buffer 2 (IB-2): 225 mM sucrose, 75 mM mannitol, 5 mM
HEPES adjusted to pH 7.4 with Tris base.
40% Percoll solution: 80 mL of Percoll dissolve in 120 mL of Isolation Buffer 1. Adjust pH to 7.4 with HCl.
24% Percoll solution: 30 mL of 40% Percoll bring to 50 mL with
Isolation Buffer 1.
15% Percoll solution: 17.5 mL of 40% Percoll bring to 50 mL with
Isolation Buffer 1.
200 mM EGTA (pH 7.4) Dissolve 7.6 g of EGTA in 80 mL of bidistilled water, adjust pH to 7.4 with KOH, bring the solution to
100 mL with de-ionized water.
Store all the reagents at 4 C.

3. Procedure
Mitochondria were isolated from different fresh brain tissues
including human and mouse. The technique can also be applied
to other species (i.e. monkey, rat, cow) (unpublished data). The
method has also been successfully used to isolate mitochondria
from fresh frozen brain tissue (unpublished data). This procedure will yield non-synaptosomal and synaptosomal (neuronal)
mitochondria. The general workow is shown in Fig. 1.
Isolation of mitochondria from human brain tissue (approx. 3 h)
(a) Put the tissue extracted during surgery in 4550 mL of ice-cold
IB-1 and place the container on ice during transport back to the
workbench.
(b) Rinse the tissue of blood by using ice-cold IB-1.
(c) Transfer the washed tissue into a beaker with fresh IB-1.
(d) Mince the tissue into small pieces using scissors to eliminate
blood trapped inside the specimen.

2.1. Reagents
Surgically extracted fresh brain tissue sample.
Sucrose (Sigma, cat. no. S0389)
Mannitol (Sigma, cat. no. M9546)
Percoll, (Sigma, cat. no. 17-0891-01)
Tris base (Sigma, cat. no. T1503)
HEPES (Sigma, cat. no. H3375)
Ethylene-bis (oxyethylenenitrilo) tetraacetic acid (EGTA; Sigma,
cat. no. E4378)

Critical step. If trapped blood in the specimen is not removed by

proper mincing, the blood products may compromise mitochondrial activity
(e) Transfer the minced tissue to the glass potter.
(f) Homogenize the tissue using 20 slow up and down strokes with
a Teon pestle operated at 400 rpm. Keep the volume of added
IB-1 5 to 10 times the estimated volume of the tissue sample.

N.K. Khattar et al. / Journal of Neuroscience Methods 263 (2016) 16

Brain tissue
(mince, remove blood)

Immuno-purification using anti-TOM22 antibody

conjugated to super-magnetic micro beads

Homogenization
with glass Teflon potter
(400rpm, 20 strokes)

Pellet (synaptosomal mitochondria)

Re-suspend and incubate with anti-TOM22
microbeads for 60 min at mitochondria
concentration of 0.3 mg/mL

Homogenate
(~30 mg/mL)

Suspension with immune- labeled mitochondria

Load onto a pre-equilibrated MACS Column
mounted on the MACS Separator

Centrifugation 1,300 g, 3 min

(repeat twice)
Supernatant

Immuno-labeled mitochondria caught in the magnetic field

Wash MACS Column mounted on the MACS
Separator 3 times

Centrifugation 21,000 g, 10 min

Pellet (Crude brain mitochondria)
Percoll step-gradient centrifugation
30,700 g 8 min

Nonsynaptosomal mitochondria
(band between 24 and 40% Percoll

Centrifugation
8000 g, 10 min

Pure immuno-labeled mitochondria caught in the magnetic field

Elute mitochondria from the MACS column

Synaptosomes
(band in the 24% Percoll)

Suspension of pure immuno-labeled mitochondria

Cell Disruption Vessel

1100 psi, 15 min

Centrifugation 13,000 g, 4 min

Pellet (Purified synaptosomal mitochondria)

Centrifugation
16,700 g, 10 min
Centrifugation
6900 g, 10 min

Crude Synaptosomal mitochondria

Pellet

Pellet

Percoll step-gradient
centrifugation
30,700 g, 10 min
Pellet

Resuspend in Isolation Buffer II

Suspension (Purified synaptosomal mitochondria)
Centrifugation 13,000 g, 4 min
Pellet (Purified synaptosomal mitochondria)

Pellet (Nonsynaptosomal or synaptosomal mitochondria)

Fig. 1. Flow chart of the mitochondrial isolation technique.

Critical step. Slow movement of the pestle during homogenization is essential to avoid disruption of the mitochondria by inducing
low pressure in homogenate during the upward movement.
Critical step. If the volume of the brain specimen is too large, then
multiple homogenizations are necessary. It is preferable to perform
all the steps to the isolation at 4 C.
(g) Depending on the nal volume, transfer the homogenate to a
16 mL or 50 mL centrifuge tube and centrifuge at 1300 g for
3 min at 4 C.
(h) Carefully decant the supernatant into an empty polycarbonate
centrifuge tube and place on ice.
(i) The pellet should be loose and be easily disrupted. Re-suspend
the pellet in 510 mL of IB-1 and centrifuge at 1300 g for 3 min
at 4 C again.
(j) Repeat steps (gi) as many times as necessary depending on the
total volume of the brain specimen.
(k) Centrifuge the pooled supernatant at 21,000 g for 10 min at
4 C.
Critical step. Make sure no loose pellet is transferred for hi-speed
centrifugation. Blood and other impurities found in the pellet may
compromise mitochondrial activity.
(l) During the centrifugation step, prepare the Percoll gradient by
creating a discontinuous layer of 1.5 mL of 40% Percoll at the
bottom of the tube with 4 mL of 24% Percoll layered on top of it.
(m) Discard the supernatant and re-suspend the pellet in 15% Percoll.
(n) Gently transfer 3.5 mL of re-suspended pellet in the 15% Percoll
on the top of 24% Percoll to create a third discontinuous layer.

(o) Centrifuge at 30,700 g for 8 min at 4 C using slow acceleration/deceleration regimes.

(p) Collect the synaptosomal fraction (SN) of the isolation between
the 15% and 24% layers.
(q) Collect the non-synaptosomal fraction (NS) of mitochondria
between the 24% and 40% layers of Percoll.
(r) Dilute the SN fraction at a 1:1 ratio with IB-1 and incubate in
the cell disruption vessel at 1000 psi for 15 min at 4 C using a
magnetic stirrer.

Critical step. Increasing the pressure above 1000 psi would result
in decreased amount/activity of the synaptosomal mitochondria.

(s) Adjust the volume of the NS fraction to 1012 mL and then

centrifuge at 16,700 g for 10 min at 4 C.
(t) Carefully aspirate the supernatant and re-suspend the loose
pellet in 10 mL of IB-1
(u) Centrifuge at 6900 g for 10 min at 4 C.
(v) Decant the supernatant and re-suspend the pellet in 1 mL of
IB-2 and centrifuge at 8000 g for 10 min at 4 C.
(w) Decant the supernatant and re-suspend the pellet. The NS
mitochondria in the collected suspension are ready to be used
for further experiments.
(x) Collect the SN fraction from the cell disruption vessel and
centrifuge at 16,700 g for 10 min at 4 C.
(y) Carefully aspirate the supernatant and re-suspend the loose
pellet in 10 mL of IB-1
(z) Centrifuge at 6900 g for 10 min at 4 C.
(aa) Decant the supernatant and re-suspend the pellet in 1 mL of
IB-2 and centrifuge at 8000 g for 10 min at 4 C.

N.K. Khattar et al. / Journal of Neuroscience Methods 263 (2016) 16

(e) Add 1 mL of IB-2 into the column reservoir and place a 1.5 mL
tube under the column nozzle. Remove the column from the
separator and ush out the mitochondria by rmly pushing the
liquid through the column using the plunger (provided with
each LC column).
(f) Pellet the mitochondria by centrifuging the eluted solution at
13,000 g for 4 min at 4 C.
Critical step. To increase the yield of mitochondria, repeat steps
E and F once or twice until the ushed liquid out of the column
becomes clear.

Fig. 2. Assessment of purity using our mitochondrial isolation method. As mentioned in the text, immunoblotting was done using 10 g of protein to compare
the purity of the mitochondrial samples obtained using both the traditional Percoll
method and our new method. WB is the sample from whole brain homogenate,
which was used as a control to the mitochondrial enrichment methods. NS (P) and
Syn (P) represent samples from non-synaptosomal and synaptosomal mitochondria respectively, isolated using the traditional Percoll method. NS (M) and Syn
(M) represent samples from non-synaptosomal and synaptosomal mitochondria,
respectively, isolated using our new MACS-based method. The samples were
resolved by 412% gradient SDSPAGE and transferred onto PVDF-FL membranes
using a wet transfer apparatus at 40 V overnight at 4 C. The membranes were then
incubated for 1 h with Odyssey blocking buffer to prevent nonspecic binding. The
PVDF-FL membrane was then incubated with primary antibodies overnight at 4 C.
After washing with phosphate-buffered saline (PBS), the membranes were further
incubated with IRDye Goat secondary antibodies at 1:30,000 dilution in Odyssey
blocking buffer at room temperature for 1 h. Following further washing with PBS,
immunoreactive protein bands were visualized on Odyssey CLx imager. The NS (P)
samples show a much smaller contamination level compared to Syn (P) samples.
Using our MACS-based method, -tubulin and synaptophysin are absent in the Syn
(M) samples, and calreticulin is signicantly decreased, which indicates that the
mitochondria are pure of other cellular contaminants.

(bb) Decant the supernatant and resuspend the pellet into minimal
volume of IB-2. The SN mitochondria are ready to be further
puried with the MACS isolation kit.
4. Purication of synaptosomal mitochondrial fraction
using MACS mitochondria isolation kit (approx. 2 h)
(a) Re-suspend the disrupted SN fraction in 10 mL freshly diluted 1
x separation buffer and incubate it on the nutator shaker (Clay
Adams, cat. no. 421105) at a speed of 12 rpm in the presence
of 75 L anti-TOM22 microbeads for 60 min at mitochondria
concentration of 0.3 mg/mL at 4 C.
(b) Mount LC column on the magnetic eld of MACS separator and
pre-equilibrate the column by washing it once with 3 mL of
1 separation buffer.
(c) Apply 3.3 mL of mitochondrial suspension into the reservoir of
LS column. Wait approximately 5 min until the liquid is ltered
out and repeat this step twice.
Critical step. To increase the yield of mitochondria it is possible
to repeat step C with ltered solution.
(d) Wash the column 3 times using 3 mL of 1 separation buffer.

(g) Wash the collected fraction of pure SN mitochondria twice

using IB-2 by centrifuging at 13,000 g for 4 min at 4 C. This
step is important to remove the separation buffer as its composition affects subsequent mitochondrial studies. Re-suspend
the mitochondria in IB-2 using 20 L of IB-2 buffer (as little
as possible). It is important that mitochondria remain in high
concentration suspension to maintain proper functionality.
(h) Transfer the mitochondrial suspension into a 1 mL test tube
and store on ice until used for functional tests, no longer than
4 h. Otherwise, the sample may be stored at 80 C or in liquid
nitrogen.
5. Results
This method is specically focused to provide highly enriched
neuronal mitochondria that exhibit good functional activity. To
ensure that the yielded mitochondria fulll these criteria, both
synaptosomal and non-synaptosomal mitochondria were tested
for purity and functionality. Avoiding the presence of BSA in
the nal mitochondrial sample improves the analysis of mitochondrial proteins. It is important to note that the isolation
of highly puried synaptosomal mitochondria requires an additional 2 h of processing as compared with non-synaptosomal
mitochondria.
5.1. Assessment of mitochondrial purity
Purity of isolated mitochondria was tested using immunoblotting against known markers for mitochondrial and cytosolic
fractions. Ten g of protein obtained from the different isolation
methods (Percoll alone or Percoll plus MACS or samples from whole
brain tissue lysate) were resolved by 412% gradient SDSPAGE
and transferred onto polyvinylidene uoride-FL (Millipore, cat. no.
IPFL-00010) membranes using a wet transfer apparatus (Bio-Rad;
Hercules, CA) at 40 V overnight at 4 C. Nonspecic antibody binding was blocked with Odyssey Blocking Buffer (Li-Cor, cat. no.
927-40000) at room temperature for 1 h. The PVDF-FL membrane
was then incubated with primary antibodies (1:1,000 dilution in
the Odyssey Blocking Buffer) at 4 C overnight. The primary antibodies used included total OXPHOS Rodent WB antibody Cocktail
(Abcam, cat. no. ab110413) which is a mixture of ve antibodies
against different components of the oxidative phosphorylation
chain: Respiratory Complex I, subunit NDUFB8 20 kDa (ab110242),
Respiratory Complex II, subunit SDHB 30 kDa (ab14714), Respiratory Complex III-Core protein 2, subunit UQCRC2 50 kDa
(ab14745), Respiratory Complex IV, COX I (ab14705) and ATPsynthase (Complex V), alpha subunit ATP5A (ab14748). Other
antibodies used were anti--Tubulin (Sigma, cat. no. T5168),
anti-Synaptophysin (Sigma, cat. no. S5768), anti-calreticulin
(Abcam, cat. no. ab2907), anti-PEX-14 (Abcam, cat. no. ab113286)
and anti-LAMP1 (Abcam, cat. no. ab108597). After washing with
phosphate-buffered saline, the membranes were further incubated with Goat anti-mouse or anti-rabbit infrared-labeled IRDye

N.K. Khattar et al. / Journal of Neuroscience Methods 263 (2016) 16

Fig. 3. Respiration of isolated mitochondria. (A) Respiration of isolated non-synaptosomal (NS) and synaptosomal (Syn) mitochondria is shown. Upper traces are representative examples of changes in O2 concentration, and lower traces correspond to the respiration rates. Sequential additions of mitochondria (mitos), respiration substrate, 5 mM
glutamate-malate (GM), 100 M ADP (ADP), oligomycin (Oligo) and 1 M FCCP is shown by arrows. (B) Test for mitochondrial preparation functional stability. Mitochondria
isolated with percoll (NS, Syn) and present method (Syn MACS) were kept on ice after isolation and the respiration was tested after indicated time periods. Numbers on
curves indicate mitochondrial protein concentration during storage on ice. Respiration was measured at protein concentration of 0.05 mg/mL.

secondary antibodies (Li-Cor) (1:30,000 dilution in Odyssey

Blocking Buffer) at room temperature for 1 h. Immunoreactive
protein bands were visualized on Odyssey CLx imager (Li-Cor)
and quantitated using the provided Image Studio software. Fig. 2
shows that Percoll-isolated synaptosomal mitochondria retain
substantial amounts of synaptophysin, a marker of synaptic vesicles and calreticulin, which is a marker of endoplasmic reticulum
contamination. Additional purication of the same Percoll-isolated
synaptosomal mitochondria with the MACS apparatus decreased
synaptophysin, calreticulin, PEX-14 (data not shown) and LAMP-1
(data not shown) contamination below detection limits. Given
the limited availability of surgically extracted human cortex,
the purity blots were limited to whole brain samples, nonsynaptosomal mitochondria and MACS-puried synaptosomal
mitochondria.
5.2. Assessment of mitochondrial functionality
Functionality of isolated mitochondria was assessed by measuring both mitochondrial respiratory function and mitochondrial
protein import.
5.3. Mitochondrial respiration
Measurements were performed using a Clark type electrode
using an OROBOROS Oxygraph-2k (Oroboros, Innsbruck, Austria)
(Gnaiger, 2001) at 30 C. Respiration of mitochondria (0.125 mg of
protein/mL for non-synaptosomal and 0.2 mg/mL for synaptosomal) was recorded in medium contained 5 mM HEPES, 125 mM
KCl, 2 mM KH2 PO4 , 5 mM MgCl2 , 0.02 mM EDTA, and 0.2 mg/mL
BSA (fatty acid free), pH 7.4. Respiratory substrates were used to
feed electrons into the respiratory chain either via complex I (5 mM
malate + 5 mM glutamate) or via complex II (5 mM succinate). The
respiratory control ratios (RCR) were calculated by dividing the
rate of respiration in the presence of respiratory substrates (resting
state respiration, V2) and the rate of respiration in the presence
of both respiratory substrates with the addition of 120 M ADP
(ADP-stimulated respiration, V3). Oligomycin was then added to
inhibit the mitochondrial ATP-synthase (Complex V). Maximum
respiratory capacity was measured in the presence of respiratory

substrates and FCCP (1 M), which is a mitochondrial respiration

uncoupler.
Fig. 3A demonstrates that isolated mitochondria responded
normally to an addition of ADP and FCCP. Respiratory control ratios (RCR) are shown on the table. Both mitochondrial
preparations yield functional mitochondria even though there
is a slight loss of respiratory capacity with the longer isolation
time.
Fig. 3B shows the respiratory control ratios (RCR) of the isolated
mitochondria over 15 h time period following the completion of
the isolation protocol. The mitochondria were kept on ice at 4 C
overnight before RCR was tested again at 15 h and showed retention
of functionality of the isolated mitochondria. It is also important to
note that higher concentration of mitochondria contributes to their
viability.
5.4. Mitochondrial import assay
Mitochondrial matrix protein import requires both presence
of the mitochondrial membrane potential (which indicates the
structural integrity of the mitochondria and the activity of the
mitochondrial respiratory chain) and functional activity of the
mitochondrial enzymes involved into the protein uptake and
processing. Thus, it is an ideal assay to measure the integrity
and functionality of isolated mitochondria. A pre-ornithine transcarbamoylase (pOTC) cDNA in pGEM-3Zf(+)-pOTC plasmid was
transcribed and translated in vitro using the TNT T7 Coupled Transcription/Translation system (Promega, cat. no. L1170)
in the presence of L-[35 S]-methionine (Perkin Elmer, cat. no.
NEG709A500UC). Following translation, [35 S]-methionine-labeled
pOTC was incubated with isolated mitochondria at 25 C for different time points as shown, and mitochondria containing imported
OTC were collected by centrifugation (9,000 g for 10 min at 4 C)
and separated using SDS-PAGE. The radioactively labeled protein
were visualized by exposing dried gel to a phospho screen (BioRad) and scanned on PMITM Personal Molecular Imager system
(Bio-Rad). Cleaved mature OTC (mOTC), which represents the completion of import into the mitochondrial matrix, and uncleaved
OTC (pOTC), were quantied using the provided software (Quantity One, version 4.6.9, Bio-Rad). Cleaved mOTC (35.2 kDa) has
a smaller molecular weight than pOTC (39.9 kDa) and therefore

N.K. Khattar et al. / Journal of Neuroscience Methods 263 (2016) 16

6. Conclusion
With this method, we demonstrate the ability to isolate highly
enriched and puried functional neuronal mitochondria from
human brain. This method represents a signicant advance over
existing methods that result in highly enriched and puried
mitochondria not specic to neurons, or neuronal mitochondrial
fractions contaminated with synaptosomal vesicles, endoplasmic
reticulum, lysosomes, peroxisomes or other cellular debris. The
use of our method allows investigators to probe both structural
and functional properties of neuronal mitochondria without interactions from other cellular contaminants. It is important to note
that our method will eliminate any mitochondria-endoplasmic
reticulum domains and therefore makes it unsuitable for any experiments studying those microdomains.
Conict of interest statement
The authors have no nancial conicts of interest to declare.
Acknowledgements
This work was supported by US National Institutes of Health
grants R01 NS039324 and NS077748 (to R.M.F.), the DSF Charitable Foundation, 132RA02 (R.M.F.), the Pittsburgh Brain Institute (to
R.M.F.), and the Copeland Fund/Pittsburgh Foundation (to S.V.B.).
References

Fig. 4. Mitochondria isolated using our method are functional. Mitochondria

isolated from extracted human cortex were subjected to pOTC import assays. Representative results of pOTC import reaction using non-synaptosomal mitochondria (NS
(P), synaptosomal mitochondria isolated the traditional Percoll method (Syn (P)) and
synaptosomal mitochondria isolated using our method (Syn (M)). Representative gel
images used for quantication are shown. Import reaction times are indicated. There
is a decrease in functionality between non-synaptosomal mitochondria and synaptosomal mitochondria regardless of the isolation technique. There is a decrease in
pOTC import in the synaptosomal mitochondria isolated using our method. This
decrease is non-signicant (60 min time point, Students t-test, p = 0.1175). The
error bars reported on this graph represent the standard error of the mean (SEM)
associated with the dataset.

migrates faster on the gel. This allowed the quantication of the

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using our method. Given that the functionality of the mitochondria
is intact, any further functional or structural studies to examine differences between neuronal and non-neuronal mitochondria can be
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