Cell Biology Protocols

2 A glass–Teflon homogenizer or juicer mixer can be used, although the spinal cords are more finely homogenized with a polytron.

3 Filter device for ultrafiltration.

4 Rat or mouse spinal cords can be used for preparation of NF. Rat or mouse spinal cords are easily ejected from an upper backbone by injecting water into the lower part of the backbone. However, the separation of NF-M and NF-L by column chromatographies is difficult in comparison with porcine or bovine NF. If you want to use rat or mouse NF-L, we recommend to express it in E. coli as described in section II).

5 Preparation of NF-M and NF-H from E. coli is not recommended, although purification of NF-H can be done. Not only is their expression low, but also a lot of truncated forms are generated particularly for NF-M.

6 NF-L is obtained as a inclusion body but is solubilized by 6 8 M urea.

7 NF-H is eluted earlier than NF-L and NF-M as a single band on the gel of SDS-PAGE, but NF-L and NF-M are eluted with a overlap in the order of NF-M and NF-L.

8 NF-L at lower concentrations (0.1 mg/ml) can assemble into filaments but higher concentrations are better to obtain longer filaments. The extended dialysis up to 24 h results in longer filaments.

PROTOCOL 6.35

341

References

1.Pant, H. C. and Veeranna (1995) Biochem. Cell Biol., 73, 575–592.

2.Julien, J. P. and Mushynski, W. E. (1998)

Prog. Nucleic Acid Res. Mol. Biol., 61, 1–23.

3. Sasaki, T.,	Taoka,	M., Ishiguro,	K.,
Uchida, A.,	Saito, T., Isobe, T. and
Hisanaga, S.	(2002)	J. Biol. Chem.,	277,

36 032–36 039.

4.Julien, J. P. and Mushynski, W. E. (1983) J. Biol. Chem., 258, 4019–4025.

5.Carpenter, S. (1968) Neurology, 18, 841–851.

6.Mersiyanova, I. V., Perepelov, A. V.,

Polyakov, A. V., Sitnikov, V. F., Dadali, E. L., Oparin, R. B., Petrin, A. N. and Evgrafov, O. V. (2000) Am. J. Hum. Genet., 67, 37–46.

7.De Jonghe, P., Mersivanova, I., Nelis, E., Del Favero, J., Martin, J. J., Van Broeckhoven, C., Evgrafov, O. and Timmerman, V. (2001) Ann. Neurol., 49, 245–249.

8.Perez-Olle, R., Leung, C. L. and Liem, R. K. (2002) J. Cell Sci., 115, 4937–4946.

9.Brownlees, J., Ackerley, S., Grierson, A. J.,

Jacobsen, N. J. O.,

Shea, K.,

Anderton,

B.H., Leigh, P. N., Shaw, C. E. and Miller,

C.C. J. (2002) Hum. Mol. Gen., 11, 2837– 2844.

10.Lavedan, C., Buchholtz, S., Nussbaum, R. L.,

Albin, R. L. and Polymeropoulos, M. H.

(2002) Neurosci. Lett., 322, 57–61.

11.Hisanaga, S., Gonda, Y., Inagaki, M., Ikai, A. and Hirokawa, N. (1990) Cell Regul., 1, 237–248.

12.Ching, G. Y. and Liem, R. K. (1993) J. Cell Biol., 122, 1323–1335.

13.Lee, M. K., Xu, Z., Wong, P. C. and Cleveland, D. W. (1993) J. Cell Biol., 122, 1337– 1350.

PROTOCOL 6.36

α-Synuclein fibril formation induced by tubulin

Kenji Ueda´ and Shin-ichi Hisanaga

Introduction					Reagents
Human α-synuclein was originally iden-					Express human α-synuclein in Escherichia
tified as the precursor protein of non-β-					coli BL21(DE3)pLysS using an expression
amyloid β component (NAC) in an amy-					vector with isopropyl-1-thio-β-D-galacto-
loid-enriched fraction from Alzheimer’s					pyranoside (IPTG) and purified by HPLC
disease brains [1]. Later, three missense					methods [3, 4].	Briefly, a soluble frac-
mutations in the α-synuclein gene were					tion of E. coli	expressing α-synuclein
discovered in certain pedigrees with auto-					was fractionated by HPLC column chro-
somal-dominant familial Parkinson’s dis-					matographies of anion exchange (DEAE
ease	and dementia with		Lewy	bod-	5PW, TOSO, Tokyo, Japan), hydropho-
ies.	Increasing	evidence	suggests	that	bic (Phenyl TOYOPEARL, TOSO), and
α-synuclein is a common pathogenic					reversed phase (TSK-gel phenyl 5PW,
molecule in several neurodegenerative dis-					TOSO) columns sequentially. From 1 l cell
eases, collectively known as synucle-					culture, 80 100 mg of α-synuclein is
inopathies. α-Synuclein is a soluble neu-					obtained with a purity of more than 98%.
ronal	protein of	unknown	function,	but	1 Purified α-synuclein can be lyophilized
is the major filamentous component of					and stored at −80 ◦C. Microtubule proteins
pathological inclusions in those diseased					can be isolated from rat or porcine brains
brains. α-Synuclein has the property of					by two cycles of a temperature-dependent
forming fibrils by itself in vitro, and muta-					assembly and disassembly according to
tions	of α-synuclein accelerate the			fib-	Shelanski et al. [5]; see also Protocol 6.34.
ril formation [2]. Furthermore, several fac-					Purify tubulin from the microtubule pro-
tors are known to accelerate fibril forma-					teins by phosphocellulose (Whatman P11)
tion in vitro. It is essential to understand					column chromatography [6] and store at
the mechanism for filament formation in					−80 ◦C. All standard laboratory reagents
neurodegenerative diseases.			Recently, it		should be of high purity and good quality.
was demonstrated that tubulin seeds α-					Milli Q water should be used.
synuclein fibril formation under physiolog-
ical conditions [3]. Here, the procedures					Equipment
for preparing proteins and fibril formation,
and the detection of fibrils are described.					Eppendorf or other laboratory tubes

PROTOCOL 6.36

343

(a)

A 400 nn

(b)

0.4α – syn + tub controls

0.3

0.2

0.1

0

0

24

48

72

96

Time (h)

Figure 6.41 Fibril formation of α-synuclein in the presence of tubulin: (a) Fibril formation of α-synuclein monitored by quasi-elastic light scattering. α-Synuclein (300 µM) in 25 µl of PBS was incubated at 37 ◦C with 1 µM of tubulin, and 1 µl of the reaction mixture was taken and measured its scattering at 400 nm at various times. The fibrils began to form after 6 h of incubation, and this formation steadily increased and reached a plateau in 3 days. An open square represents four kinds of controls that were completely flat. Controls: (1) 300 µM α-synuclein; (2) 1 µM tubulin; (3) 300 µM α-synuclein+1 µM BSA; (4) 300 µM BSA + 1 µM tubulin. The value represents the average of three determinations. (b) A negative staining electron micrograph of representative α-synuclein fibrils. A number of long, 10-nm-wide filaments are formed. Bar = 100 nm

37 ◦C water bath

Materials for preparation of negatively stained EM specimens (see Protocol 2.4 ) Microcentrifuge

Spectrophotometer equipped with a ultramicro amount sample cell (we use a Hitachi Gene Spec III, which has enabled ultra-micro volume quantitative analysis, using as little as 1 µl)

Transmission electron microscope

4.Monitor fibril formation by light scattering at OD 400 nm using 1 µl of the

reaction mixture with a Gene Spec III spectrophotometer. 3 4 5

5.Prepare EM specimens negatively stained with 2% uranyl acetate.

6.Assess the fibril formation on specimen grids by an EM.

An example of fibril formation by α- synuclein is shown in Figure 6.41.

Procedure

1. Thaw α-synuclein and tubulin stored at −80 ◦C quickly in a water bath and keep them on ice. 2

2.Add ice-cold sterilized PBS, pH 7.4 to make 300 µM solution of α-synuclein with 1 µM of tubulin in 25 µl.

3.Incubate at 37 ◦C without agitation for 24 h (or longer, as required). Fibrils begin to form after 6 h of incubation.

Notes

1 Purified recombinant α-synuclein is unstructured, heat-stable, highly soluble, and exists as a monomer in an aqueous solution.

2 Tubulin is an unstable protein, losing its polymerization activity upon keeping it on ice for long time. Tubulin should be stored in aliquots to avoid repeated freezing/thawing and thawed freshly on each experimental day.

344 IN VITRO TECHNIQUES

3 Take 1 µl aliquots from the incubation mixture into an ultra-micro sample cell without agitation at the time of monitoring.

4 α-Synuclein can undergo self-aggre- gation under certain conditions, such as increasing time lag, high temperature, high concentration and low pH.

5 Light scattering intensity indicates the total mass of fibrils formed.

References

1. Ueda,´ K., Fukushima, H., Masliah, E., Xia, Y., Iwai, A., Yoshimoto, M., Otero, D. A. C.,

Kondo, J., Ihara, Y. and Saitoh, T. (1993) Proc. Nat. Acad. Sci. USA, 90, 11 282–11 286.

2.Ma, Q. L., Chan, P., Yoshii, M. and Ueda,´ K. (2003) J. Alzheimer Dis., 5, 139–148

3.Alim, M. A., Hossain, M. S., Arima, K., Takeda, K., Izumiyama, Y., Nakamura, M., Kaji, H., Shinoda, T., Hisanaga, S. and Ueda,´ K. (2002)

J.Biol. Chem., 277, 2112–2117.

4.Hossain, S., Alim, A., Takeda, K., Kaji, H., Shinoda, T. and Ueda,´ K. (2001) J. Alzheimer Dis., 3, 577–584.

5.Shelanski, M. L., Gaskin, F. and Cantor, C. R. (1973) Proc. Nat. Acad. Sci. USA, 70, 765–768.

6.Weingarten, M. D., Lockwood, A. H., Hwo,

S.Y. and Kirschner, M. W. (1975) Proc. Nat. Acad. Sci. USA, 72, 1858–1862.

PROTOCOL 6.37

Amyloid-β fibril formation in vitro

J. Robin Harris

Introduction

Fibrillogenesis of the amyloid-β peptide (Aβ) and inhibition of fibrillogenesis is of considerable interest for biomedical scientists and clinicians concerned with the development and prevention of Alzheimer’s disease. The Aβ peptide, proteolytically cleaved in vivo from the soluble amyloid precursor protein, exists as a range of peptide length, between 39 and 44 amino acids. The 42 amino acid peptide (Aβ1−42) is believed to be the predominant amyloid peptide incorporated in fibril form within Alzheimer senile plaques [1]. Although biophysical (e.g. spectroscopy and atomic force microscopy), and biochemical studies (e.g. thioflavin-T fluorescence, Congo red binding) have been used to assess Aβ fibrillogenesis [2], TEM is the most rapid and direct procedure, and it enables comment to be made on the progression of different oligomerization, protofibril and fibril states, and the interaction of fibrils with other proteins and physiological reagents such as cholesterol and sphingomyelin [3].

Reagents

Synthetic amyloid-β (Aβ) peptide (1–42) (human, mouse or rat sequence, and synthetic equivalents of Aβ mutants), is available from most biochemical suppliers, often as the trifluoroacetate salt. Store at −20 ◦C, or below.

Chemicals that potentiate (e.g. cholesterol, ganglioside GM1, metal ions) or inhibit (e.g. aspirin, nicotine, vitamin E) fibrillogenesis can also be incorporated within this system, as required.

All standard laboratory reagents should be of high purity and good quality deionized water used. 1

Equipment

Incubator (37 ◦C), with shaker

Microcentrifuge or other laboratory tubes

Microcentrifuge

Materials for preparation of negatively stained EM specimens (see Protocol 2.4 )

Spectrometer/fluorescence spectrometer (if biochemical quantitation of fibrillogenesis is required)

Access to a transmission electron microscope (TEM)

Procedure

1.Warm the lyophilized Aβ peptide to room temperature before opening.

2.Add ice-cold deionized water (or buffer at defined pH) to produce a 0.2 mM/1 mg/ml solution of Aβ peptide. Keep on ice.

3.Disperse and solubilize the peptide by repeated gentle pipetting for 2–5 min.

Remove any insoluble aggregates by centrifugation, if necessary. 2

346 IN VITRO TECHNIQUES

(a)	(b)
Figure 6.42	Example of human Aβ1−42 peptide following 24 h incubation at 37 ◦C in the absence

(a) and presence (b) of cholesterol microcrystals. Note that in (a) there is a background spread of oligomeric Aβ particles, alongside short and longer A fibrils. In (b) all the oligomeric particles have polymerized to generate single and clustered fibrils which also bind to the cholesterol microcrystals. Negatively stained with 2% uranyl acetate. The scale bars indicate 200 nm (JRH, previously unpublished data)

4.Aliquot the Aβ solution into several

tubes containing an equal volume containing differing additives. 3 4

5.Incubate at 37 ◦C with constant oscillation for 24 h (or longer, as required).

6.Prepare EM specimens negatively stained with 2% uranyl acetate (or other suitable negative stain [4].

7.Assess the formation of protofibrils and helical fibrils by studying specimen grids in a TEM.

8.Aβ fibril formation can also be assessed by light microscopy/spectroscopy to determine Congo red binding or by thioflavin-T fluorescence, both of which indicate alpha-helix to beta-sheet conversion [2].

A representative example of Aβ1−42 fibrillogenesis is shown in Figure 6.42.

Notes

This procedure will take approximately 24 h.

1 Use HPLC grade, deoxygenated water. Metal ion contamination (e.g. Cu, Zn, Fe, Al) should be avoided.

2 This will necessitate determination of the protein concentration of the supernatant.

3 That is, water/buffer alone, plus and appropriate concentration of a compound that may potentiate (cholesterol, sphingomyelin, gangliosides, multivalent metal ions) or inhibit Aβ fibrillogenesis (e.g. vitamin E, Aβ fragments, apolipoprotein E4, catalase, aspirin, nicotine, Congo red and other drugs) [3, 5].

4 In view of the potentially hazardous nature of amyloid peptides and the

			PROTOCOL 6.37	347
various additives or drugs used in this	2.	Nybo, M.,	Svegag, S.-E. and Nielsen, E. H.
protocol, if injested, appropriate care		(1999) Scan. J. Immunol., 49, 219–223.
should be taken throughout.	3.	Harris, J. R. (2002) Micron, 33, 609–626.
	4.	Harris, J. R. and Scheffler, D. (2002) Micron,
		33, 461–480.
References	5.	Ono, K.,	Hasegawa, K., Yamada, M.	and
		Naiki, H. (2002) Biol. Psychiatry, 52, 880–886.

1. Rochenstein, E., Mallory, M., Mante, M.,

Sisk, A. and Masloaja, E. (2001) J. Neurosci.

Res., 66, 573–582.

PROTOCOL 6.38

Soluble Aβ1 – 42 peptide induces tau hyperphosphorylation in vitro

Terrence Town and Jun Tan

Introduction

Alzheimer’s disease (AD) has two key pathologies: the presence of neurofibrillary tangles (NFTs) and deposition of amyloid- β peptide (Aβ) as senile plaque. NFTs are predominately comprised of hyperphosphorylated tau protein, assembled primarily in the paired helical filament (PHF) conformation [1, 2]. While the mechanistic link between tau hyperphosphorylation and NFTs is not well understood, it is generally thought that increased phosphorylation promotes PHFs, resulting in disruption of the physiological role of tau in stabilizing neuronal microtubules that are necessary for axoplasmic flow [3]. Tau appears to be hyperphosphorylated at specific epitopes (particularly in the proline-rich region that contains several serine/threonine-proline motifs) in AD brain [2].

One key question concerning tau hyperphosphorylation is whether or not it is promoted by Aβ. It is now generally accepted that Aβ directly promotes increased levels of intracellular Ca2+ in neurons, thereby causing neuronal injury and apoptosis [4]. Recent evidence has established the signalling cascade that promotes neuronal cyclin-dependent kinase 5 (Cdk5) activation induced by p25, with the first event being increased intracellular Ca2+ levels, which then promotes calpain activation, which, in turn, goes on to directly

cleave p35 to p25 [5]. Excess p25 then complexes with Cdk5, thereby boosting its activity, an event that is associated with tau hyperphosphorylation at ADspecific phosphoepitopes and, not surprisingly, neurotoxicity [5, 6]. Further, as studies of human biopsies and aged canine and primate brains have consistently shown that dystrophic neurites precede deposition of Aβ in the formation of neuritic plaques [7–10], the putative role of soluble forms of Aβ peptide per se in promotion of tau phosphorylation is of interest. We have recently demonstrated that, in p35-overexpressing neuron-like cells, soluble Aβ is a potent activator of the Cdk5/p25 pathway, leading AD-like tau phosphorylation in vitro [11].

Reagents

1.The p35-overexpressing N2a cell line is required for these experiments and is available from our laboratory upon request. These cells are grown in Eagle’s minimum essential media sup-

plemented with 2 mM L-glutamine and Earle’s balanced salt solution adjusted to contain 1.5 g/l sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 10% fetal bovine serum and 400 µg/ml of G418, and are induced to differentiate into neuron-like cells by serum withdrawal

and the addition of 0.3 mM dibutyryl cAMP for 48 h.

2.To examine phospho-tau and total tau levels, the following anti-tau antibodies are needed: AT8 (Innogenetics, Norcrosis, GA), AT270 (Innogenetics), pS199 (BioSource International, Inc. Camarillo, CA), and pS396 (BioSource International, Inc.) against phosphorylated forms of tau, and C-17 (Santa Cruz Biotechnology, Santa Cruz, CA) against total tau protein.

3.Synthetic human Aβ1 – 42 peptide is available from QCB Biosource International (Camarillo, CA) and other suppliers.

4.To perform Western immunoblotting, the following reagents are needed in addition to the above-mentioned primary antibodies: buffers [cell lysis

buffer containing phosphatase and protease inhibitors supplied as 10× from NEB, Beverly, MA; Tris-buffered saline (TBS), Tris/glycine/SDS buffer, laemmli sample buffer, and blocking and antibody dilution buffer consisting of TBS with 5% w/v non-fat dry milk and 0.05% Tween-20, obtained from Bio-Rad]; 10% ready gels (Bio-Rad); Hybond PVDF membranes (Bio-Rad); appropriate secondary antibodies conjugated with horseradish peroxidase (HRP) and luminol reagent (Santa Cruz Biotechnology).

5.To perform immunocytochemistry, a cell permeabilizing solution of 0.05% v/v Triton X-100 diluted in complete

medium described above is needed, and a phosphate-buffered saline (PBS) rinse solution, a fixing solution of 4% w/v paraformaldehyde in PBS, serum-free blocking solution (Dako, Carpinteria, CA), and the LSAB + immunochemistry kit (Dako, Carpinteria, CA) are needed.

PROTOCOL 6.38

349

Equipment

Immunoblot Apparatus

Fluor-S MultiImager with Quantity One software (Bio-Rad), for densitometric analysis of protein bands

Freezer −80 ◦C

Inverted light microscope, for tissue culture observation

Light microscope, preferably with a digitizing camera system

Microcentrifuge tubes

Microcentrifuge, 4 ◦C

Protein gel electrophoresis system

Tissue culture CO2 incubators, for growing cell cultures

Spectrophotometer

Procedure

Preparation of Aβ1 – 42 peptide

Add Sigma water to human Aβ1 – 42 peptide to a concentration of 250 µM immediately before all experiments. This stock solution can then be diluted to 1, 3 or 5 µM in cell culture media for tau phosphorylation experiments. Based on our recent data [11], more than 95% of the Aβ1 – 42 employed in this experimental paradigm (including all doses studied, under both cell-free and cell-present conditions) remains in soluble form throughout the experiments.

Cell culture conditions

1.Plate p35-overexpressing N2a cells at 5 × 105 cells/well in six-well tissue culture plates (for Western immunoblotting analysis of phospho-tau), or plate on glass coverslip inserts in six-well tissue culture plates (for immunocytochemistry of phospho-tau) in complete media described above.

2.To differentiate these cells, subject p35-overexpressing N2a cells to serum

350 IN VITRO TECHNIQUES

(a)

		Abeta Abeta	Abeta		Abeta	Abeta Abeta
Control1	microMmicroMmicroM		Control1	microMmicroMmicroM
	3	5		3	5

-phospho-tau

-total tau

N2a/p35 cells

N2a/mock

	(b)
	ratio	1	Control
		0.8	Abeta1-42 (1 microM)
	tau		Abeta1-42 (3 microM)
		0.4	Abeta1-42 (5 microM)
	tau/total
		0.6
	-
	phospho	0.2
		0
		N2a/p35 cells	N2a/mock

Figure 6.43 Treatment of p35-overexpressing N2a cells with Aβ1 – 42 results in increased tau phosphorylation by Western blot. p35 or mock-transfected N2a cells were differentiated and treated with a dose range of Aβ1 – 42 (1, 3 or 5 µM Aβ) or went untreated (control) for 24 h. After incubation, phospho-tau epitopes were detected by Western blot and were normalized to total tau protein (as detected by anti-tau antibody C-17). (a) membranes were probed with AT8 (above) or C-17 (below), and representative blots illustrate an Aβ1 – 42 dose–response relationship only in p35-transfected N2a cells. (b) densitometric analysis (ratio of phospho-tau to total tau signal ±1 SEM, n = 3 for each condition presented) for pooled results from (A) reveals significant treatment effects of Aβ1 – 42 dose on p35-transfected N2a cells (P < 0.001) but not on mock-transfected cells (P > 0.05)

withdrawal and give 0.3 mM dibutyryl cAMP for 48 h.

3.These cells then remain untreated (as a control), or are treated with a dose range of Aβ1 – 42 (1, 3 or 5 µM) for 24 h.

Western immunoblotting analysis of tau hyperphosphorylation

1.Preparation of cell lysate:

(a)Wash treated N2a cells twice in the six-well plate with ice-cold PBS and drain off the PBS.

(b)Add ice-cold lysis buffer (containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol- phosphate, 1 mM Na3VO4, 1 µg/ml

leupetin and 1 mM PMSF) at a volume of 0.1 ml lysis buffer per 1 × 106 cells.

(c)Scrape these cells off the plate with a plastic cell scraper and transfer the lysate into a microcentrifuge tube.

Gently rock the suspension on a rocker in the cold room for 15 min to lyse the cells.

(d) Centrifuge the lysate at 14 000g in

a pre-cooled centrifuge (4 ◦C)	for
15 min. Immediately transfer	the

supernatant to a fresh centrifuge tube.

(e)Quantify the total protein content of supernatants for each sample using the Bio-Rad protein assay kit and aliquot the sample into

several tubes and keep these tubes at −80 ◦C.

2.Western immunoblotting:

(a)Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on cell lysates (50 µg of total protein of each sample) and transfer electrophoretically to Hybond PVDF membranes.

(b)Wash the Hybond PVDF membrane twice with dH2O and mark the transferred side of membrane with a pencil.