Cultures

Immortalized cell lines

Immortalized cell lines display the advantages of cell culture models to the greatest extent. They represent homogenous populations of continuously proliferating cells. This allows large-scale experiments with reproducible results in a variety of tests. Genetic material can be brought into the cell effectively by chemical and physical means and cells can be frozen at —150° C. This allows to generate banks of cell lines, stably expressing various proteins of interest. Overexpression of a newly identified disease-causing gene or if a new mutation within a known gene is commonly the first step in its characterization since various biochemical and molecular studies can be readily performed. Freezing aliquots of low passage cell stocks is crucial to reproduce results since even cell lines are known change their appearance and behaviour with increasing passages as they accumulate genetic and epigenetic alterations. This effect, known as ''replicative senescence'', can

Table 1. Culture systems of dopaminergic neurons

Name

Origin

Neuronal differentiation

Dopaminergic phenotype1

a-Synuclein pathology

SH-SY5Y

human

retinoic acid

DAT, D2R, DA, TH,

eosinophilic cytoplasmic

neuroblastoma

±BDNF

vesicles, DA release2

inclusions upon co-overexpression of a-synuclein and synphilin-13

PC12

rat

NGF

DAT, D2R, DA,

synuclein is upregulated

neuroblastoma

TH, vesicles, DA release4

following NGF5; cytoplasmic inclusions

upon proteasome inhibition6 and

methamphetamine7

MN9D

mouse hybrid

GDNF, retinoic

DA, TH, D2R. no DAT9

no record found

mesencephalic

acid, overexpression

but sensitivity to low

cells

of Nurr18

doses of MPP+10

CSM14.111

rat embryonal mesencephalon

39° (temperature-

sensitive immortalization)

TH

no record found

MESC2.1012

8 week old

tetracycline,

TH, DAT, DA

express endogenous

human

GDNF, dibutyryl

release, electrically

human WT a-synuclein,

mesencephalon

cyclic AMP

active

inclusions following overexpression of synuclein and administration of amphetamine

primary

E13 rodent

without special

5-10% dopaminergic

endogenous synuclein-113;

midbrain

embryo

treatment

neurons

inclusions following

cultures

midbrain

proteasome inhibition14

organotypic

postnatal rat

already

postnatal dopaminergic

no record found

slice cultures

substantia nigra pars compacta

differentiated

neurons in their natural environment

Abbreviations: DAT Dopamine transporter, D2R D2 dopamine receptor, DA dopamine, TH tyrosine hydroxylase; 2Presgraves et al. (2004a, b); 3Marx et al. (2003); Smith et al. (2005); 4Pothos et al. (2000); Beaujean et al. (2003); 5Stefanis et al. (2001); 6Rideout et al. (2001); 7Fornai et al. (2004); transcription factor that is active in dopaminergic neurons; 9Hermanson et al. (2003); 10Kim et al. (2001); 11Haas and Wree (2002); 12Lotharius et al. (2002); 13Rat homologue of human alpha-synuclein; 14McNaught et al. (2002)

make it hard to reproduce results generated with one special batch of cells. Another drawback of proliferating cells is the difficulty to differentiate between growth arrest and real cell death as both ultimately result in a difference in cell number between "treated" and ''untreated'' cells. In our hands, SH-SY5Y exposed to 250 mM 1-methyl-4-phenylpyri-dium (MPP+) displayed only growth arrest after 48 and actual cell death not before 72h (Soldner et al., 1999). We therefore encourage to establish the occurrence of cell death in each paradigm using a morphological marker (see below) before drawing conclusions from biochemical data or overall cell numbers.

As researchers in Parkinson's disease pathogenesis we are interested in the survival of mature dopaminergic neurons of the human substantia nigra pars compacta (SNc). Major characteristics of some widely used or upcoming ''dopaminergic'' cell lines are summarized in Table 1, assuming that neuronal differentiation, dopaminergic phenotype and the ability to form aggregates of alpha-synuclein - the histological hallmark of Parkinson's disease - are relevant traits to model PD. It is important to keep in mind, however, that even midbrain dopaminergic neurons are not a homogeneous population. Neurons in the SNc are much more strongly affected in PD patients and more vulnerable to MPP+ toxicity than neurons in the ventral tegmental area (VTA). Gene expression profiles of these two populations have been performed recently (Chung et al., 2005) and may indicate some further susceptibility traits specific for PD.

All of the immortalized cell lines listed in Table 1 can be differentiated to a more neuron-like phenotype including cellular processes. Neuroblastoma cells can be differentiated by retinoic acid and/or growth factors. CSM14.1 cells have been immortalized by a temperature-sensitive SV40 large T antigen. These cells differentiate at the imperimissive temperature of 39°C. Similarly, MESC2.10 cells have been immortalized with a LINX v-myc retroviral vector under control of a tet-system, allowing differentiation by tetracycline. Neuronal differentiation and in particular the growth of neuronal processes is an important feature to make a better model for neurodegenerative diseases. We and others have shown that neurotoxins such as MPP+ damage neurites long before they kill neuronal somata and therapeutic strategies may vary greatly in their effect on neuri-tic integrity and functional parameters on one hand and survival of the cell body on the other hand (Herkenham et al., 1991; von Coelln et al., 2001; Berliocchi et al., 2005). After differentiation, however, cells are generally more delicate and gene transfer is made more difficult, often leaving viral vectors as the only possibility. They are also no longer amenable to FACS sorting (see below) and some other techniques requiring cell suspensions.

Primary immature dopaminergic neurons

Primary mesencephalic cultures are typically prepared from E13 rouse or rat embryos. They contain midbrain dopamine neurons cultured in the context of their physiological neighbours. As other primary neuronal cultures, neurons readily differentiate and form neurites and synapses. Even though these cultures are often referred to as ''primary dopaminergic neurons'', TH positive neurons actually make up only 5 to 10 percent of the total population. This constitutes an often invincible obstacle for viability assays and even more for many biochemical assays because significant changes that may occur in dopaminergic neurons are diluted or cancelled by changes in non-dopaminergic neurons. Moreover, there is more variability between cultures prepared on different days and by different investigators than in cell lines due to variations in preparation and processing of the tissue.

Gene transfer in primary neuronal cultures is more difficult than in cell lines. Calcium phosphate transfection works in some cases; in most cases viral gene transfer has to be used. In our hands, lentiviruses have been successfully used whereas adenoviruses and adeno-associated viruses infect GABAergic but not dopaminergic neurons in primary mid-brain cultures.

Help for the use of primary mesencepha-lic cultures may come from the growing field of functional and automated fluorescence microscopy (for a recent review see Bunt and Wouters, 2004). These techniques will eventually allow to specifically follow individual cells in culture, determine on a single cell level activation of second messenger cascades or protein aggregation - features that thus far relied on western blots and other ''bulk'' techniques. Once this has been accomplished, the low percentage of dopami-nergic neurons in primary midbrain cultures and the often low transfection efficacy in these cultures will not be a problem for productive research on these neurons anymore.

In all cellular models, neurons are evidently deprived of their natural environment, of afferent and efferent connections. As these connections are an integral part of a neuron's life in the brain, this is no trivial change. Even though cultured cell do engage in cellular contacts, these are two dimensional only. One side of the cell is usually occupied by the glass or plastic support and another side by the liquid cell culture medium. Much of the ''natural'' environment is substituted by this culture medium. Consequently, the exact culture conditions largely determine the survival of specific types of cells. For example, primary cultures are almost purely neuronal when plated without serum. With serum, there is glial growth as well, helping the survival of neurons but complicating biochemical and viability assays even further -unless cocultures of neurons and glia are intended to study their reciprocal interactions. Another critical factor in primary cultures is cell density with cells plated at a higher density surviving better (Falkenburger and Schulz, unpublished observation) and being more resistant to serum withdrawal (Collier et al., 2003).

Organotypic cultures

Some of the drawbacks just mentioned may be avoided by culturing not dissociated neurons but brain slices (Sherer et al., 2003b; Jakobsen et al., 2005). Typically, brain slices from postnatal rat pups are used, cultured either on coverslips in ''roller tubes'' as originally described by Gahwiler (1988) or on membranes at the interface between medium and the air of the incubator (Stoppini et al., 1991). Organotypic cultures combine features of cell culture and intact animals: Neurons are more easily accessible for pharmacology, gene transfer and -most importantly - repeated imaging than inside the intact animal. Gene transfer has to rely on viral vectors, however, which are either injected to locally in the slice or added to the culture medium (for a recent review see Teschemacher et al., 2005). By choosing appropriate slicing planes, important neuronal projection (such as the nigrostratal tract) can be preserved in the culture, in addition to the local neuron-glia microenvironment. Moreover, postnatal neurons are more fully differentiated than embryonic tissue used for primary cell cultures. Similar as in primary dopaminergic cultures, ''bulk'' techniques to determine viability and biochemical alterations are generally difficult, measuring do-pamine production can be useful in some paradigms. It may be expected that with the advancement of imaging techniques such as two-photon microscopy this culture will gain relevance for some applications.

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