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The Morbus Parkinson Puzzle

Single-cell Analysis After Laser Microdissection

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After Morbus (M.) Alzheimer, M. Parkinson is the second most common progressive neurodegenerative disease. Before the first symptoms manifest themselves, up to 70 percent of dopamine-releasing neurons in the mid-brain have already died. Dr. biol. hum. Falk Schlaudraff (Institute of General Physiology of the University of Ulm, Germany) used modern laser microdissection methods to isolate and analyze cells from post mortem tissue specimens taken from M. Parkinson patients in order to gain molecular insight into the disease.

M. Parkinson research

Working as a doctoral candidate under the supervision of Prof. Dr. Birgit Liss in the Molecular Neurophysiology team at the Institute of Applied Physiology, M25, University of Ulm, Dr. biol. hum. Falk Schlaudraff researched Morbus Parkinson at the molecular level. As part of his Ph.D. he analyzed the gene expression of human dopaminergic neurons, which he isolated from post mortem midbrain tissue of Morbus Parkinson patients and control subjects using a laser microdissection system from Leica Microsystems.

A characteristic sign of M. Parkinson is the deterioration of dopaminergic neurons in the mid-brain, specifically in the substantia nigra (SN, black substance). Different causes and forms of this disease have been identified. In the case of genetic familial forms, for example, it has been possible to identify various genes that have a causal influence for M. Parkinson. However, we still don’t know whether all the relevant genes have been identified, or exactly how they contribute to pathogenesis.

There are several theories on how the disease originates. M. Parkinson can be compared to a puzzle. We have already found many of the pieces and can put some of them together, but we don’t know what the whole picture looks like. We haven’t worked out the significance of some of the pieces yet, neither do we know how many pieces of the puzzle we still have to find before we can present a full picture of M. Parkinson and actually understand the disease.

I concentrated on gene expression analysis of individual dopaminergic midbrain neurons of the substantia nigra. These cells selectively degenerate as he M. Parkinson disease progresses. Once a patient notices the cardinal symptoms of M. Parkinson, such as the resting tremor that is a characteristic of this disease, more than 70 percent of dopaminergic SN neurons have already died.

One of the aims of my research was to develop optimized qPCR (quantitative polymerase chain reaction)-based methods to enable valid comparison of the gene expression of promising gene candidates in individual neurons from human post mortem tissue from M. Parkinson patients with the gene expression of the same neurons from healthy control subjects. We developed a qPCR-based platform that could be used to isolate individual cells from native tissue and obtain highly comparable gene expression analyses. Our analysis, for example the RNA quality of the specimens, showed that the quality of the results is not influenced by the staining process and laser microdissection.

Experimental procedure

Fig. 1: Flow-chart of experimental procedure. Flow-chart representing the experimental procedure of the protocol for UV-LMD and quantitative RT–PCR gene-expression analysis of individual SN DA neurons from human postmortem PD brains and controls. Published in: Nucleic Acids Research 1–16 (2008); doi:10.1093/nar/gkn084, Copyright: Oxford University Press.

Fig. 1: Flow-chart of experimental procedure. Flow-chart representing the experimental procedure of the protocol for UV-LMD and quantitative RT–PCR gene-expression analysis of individual SN DA neurons from human postmortem PD brains and controls. Published in: Nucleic Acids Research 1–16 (2008); doi:10.1093/nar/gkn084, Copyright: Oxford University Press.

Dopaminergic neurons in substantia nigra

Analyses of the different post mortem specimens are like snapshots in a specific stage of M. Parkinson. I can compare these snapshots with each other. But I can’t use these specimens to detect whether a changed gene expression is the result or the cause of the disease. We need to do further research on this subject. 

I detected an orchestrated change of gene expression in the selective dopaminergic neurons of M. Parkinson patients. This change affects genes that are involved in the regulation of the dopamine metabolism as well as genes that code for ion channels. We showed, for example, a higher expression of several genes involved in the synthesis and provision of dopamine in the surviving dopaminergic neurons. We also looked at various gene expression patterns of ion channels that regulate the activity of the dopaminergic neurons. Here too, we noticed a change in the expression of some of the examined genes of M. Parkinson patients.

Fig. 2a: UV-LMD of individual neurons. (A) Left panel: cresylviolet (CV)-stained mouse coronal midbrain section indicating the area of the substantia nigra pars compacta (SNpc) before (left) and after (right) UV-laser-microdissection of the entire area. Right panel: gel electrophoresis results of qualitative reverse transcription (RT) multiplex-nested PCR products for a whole LMD SNpc. RT–PCR signals for all eight different dopaminergic (TH, DAT, Girk2, D2s/l) and nondopaminergic (GAD, Girk1, GFAP, CB) marker-genes, detected in the heterogeneous cellular mixture of dopaminergic, GABAergic and glial cells. (DNA-ladder: 100-bp marker).

Fig. 2b: Top: CV-stained coronal midbrain section after UV-lasermicrodissection of 15 individual DA neurons. Middle: selection and laser-microdissection of an individual neuron from upper section. Lower: after UV-LMD of individual neuron section (left) and cap-control (right) demonstrating successful isolation.
Fig. 2c: PCR products after gel electrophoresis of qualitative RT multiplex-nested PCR for individual LMD SN DA neurons and small SN DA pools. Upper and second panel: similar gene expression profile of dopaminergic marker genes (TH, DAT, Girk2, D2; not Girk2, GAD67, GFAP) in pools of 20 and single SN DA neurons illustrate sensitivity and specificity of the protocol. Third panel: expression profile of an individual calbindin-positive CB (+) SN DA neuron (D and E) Quantitative real-time RT–PCR analysis of Eno2 gene-expression using 10% of cDNA from pools of 15 mouse SN DA neurons as template. Neurons were UV-LMD-collected from either fresh or stored (1-week) and re-used tissue slices.
Fig. 2d: Representative real-time PCR traces testing for Eno2 expression, relative fluorescence levels on a logarithmic scale are plotted against PCR cycles (Δ RN: relative fluorescence, normalized to internal fluorescence marker ROX); threshold-line for determinations of threshold cycles (Ct) indicated by the green line (Δ RN 0.4).
Fig. 2e: No significant difference n.s. between Eno2 detection levels (Ct) in SN DA cell-pools from fresh and frozen tissue sections (fresh: 34.43 +/- 0.51, n=10; re-used: 34.62 +/- 0.48 n=9; p=0.785). Published in: Nucleic Acids Research, 2008, 1–16 doi:10.1093/nar/gkn084, Copyright: Oxford University Press

The impact of laser microdissection

In the last few years there have been many studies comparing complete tissue specimens of substantia nigra of M. Parkinson patients with healthy tissue. However, this comparison is misleading, as in these patients, 70 percent of the neurons that are obviously involved in the disease are already degenerated at its onset. Also, the composition and the sectioning of the examined brain tissue are extremely heterogeneous. So the “tissue” approach we have done up to now is like comparing apples with pears.

We wanted to selectively view the midbrain dopaminergic neurons that are involved in the pathogenic process and used laser microdissection for validated comparison at the single-cell level. This technique makes it possible to accurately cut individual dopaminergic neurons out of complex tissue, without contact or contamination, and analyze the gene expression in individual cells. The most prevalent type of tissue in the brain is supporting tissue: Glial cells are ten to 50 times more common than the neurons we are interested in. Without laser microdissection, it would be almost impossible to clearly characterize the relatively rare nerve cells on a molecular level; they would not be distinguishable from background noise.

The analysis of single cells frequently leads to different results from those obtained from a complete tissue examination. Studies have shown that the expression of certain microRNAs is changed in the tissue of M. Parkinson patients. We followed up one of these statements and at first we were able to confirm the results for the whole tissue. However, we also examined microdissected cells in parallel. Here we found that the microRNA expression is not changed on a single cell level. This tissue artifact was detected with the aid of laser microdissection.

We use a laser microdissection system, which enables contact-free dissection of single cells or, if necessary, larger areas of tissue. The dissected material is captured in the cap of a tube and can be processed immediately.

Fig. 3: LMD and mRNA-expression analysis of individual SN DA neurons from human PD and control postmortem brains. (A and B) Pools of neuromelanin-positive [NM(+)] neurons were isolated via LMD of cresylviolet-stained horizontal midbrain cryosections from PD (A) and control brains (B). Upper panel: PD (A) and control (B) cryosections after LMD of small pools of NM+ neurons from SNpc. Lower panels: Representative PD (A) and control (b) SNpc NM(+) neurons before (left) and after dissection (right). Insert: cap control after UV-LMD. Scale bars: 250 mm, 20 mm, respectively. (C) Scatter plot of a-synuclein gene-expression-levels in PD and control brains. a-Synuclein gene-expression of each pool of 15 NM(+) and TH(+) SNpc neurons is given as pg-equivalents of total cDNA derived from human SN-tissue per cell (standard curve quantification), determined via quantitative real-time PCR. Bars represent mean a-synuclein expression for SNpc pools of each brain_SEM. (D) Plot of the mean a-synuclein cDNA levels (_SEM) against the RNA integrity number for each brain. Brain Bank codes are indicated next to each dot (see Figure 5C and Table 1). (E) Linear regression between mean a-synuclein expression and RNA integrity number for all individual analyzed control and PD brains showed no positive correlation between higher RNA quality of the tissue and detected a-synuclein expression-levels (controls: black dotted line, R2=0.0506; PD brains: red dotted line, R2=0.9950; all analyzed brains combined: black line, R2=0.4369). Please note that PD brains showed a strong inverse correlation between RNA-integrity and detected a-synuclein expression-levels (red dotted line, R2=0.9950). (F) Mean expression levels of a-SYN, TH and ENO2 were significantly higher in individual NM(+) SN DA neurons from PD brains compared to controls (see Tables 1 and 2). Published in: Nucleic Acids Research  1–16 (2008); doi:10.1093/nar/gkn084, Copyright: Oxford University Press.

Future challenges in M. Parkinson research

At present, there are no tests for diagnosing M. Parkinson at an early stage, and there are many variants of the disease and diseases with similar symptoms. A certain percentage of M. Parkinson cases are therefore diagnosed wrongly or not diagnosed at all.

The prerequisite for successful treatment of M. Parkinson would be effective early diagnosis. If the disease could be diagnosed at an initial stage in which the neurons are just beginning to degenerate, it might be possible to prevent progressive neuron degeneration so that the disease does not break out at all. 

The identification of biomarkers in blood or cerebral fluid is currently a major research focus. There are genes that are not only expressed in the brain, but ubiquitously – in all cells. If the expression of these genes were changed in the dopaminergic neurons of an M. Parkinson case, it would be possible to examine more easily accessible tissue for diagnostic purposes. The first steps have been taken, but there’s still a long way to go before such a test can be implemented, and further long-term studies need to be conducted.

References

  1. Schlaudraff F, Gründemann J, Fauler M, Dragecivic E, Hardy J, and Liss B: Orchestrated increase of dopamine and PARK mRNAs but not miR-133b in dopamine neurons in Parkinson’s disease. Neurobiology of Aging, 22 March 2014. http://dx.doi.org/10.1016/j.neurobiolaging.2014.03.016
  2. Schlaudraff, Falk: Quantitative Genexpressionsanalysen humaner dopaminerger Neuron nach Lasermikrodissektion aus post-mortem Mittelhirngewebe von Morbus Parkinson Patienten und Kontrollen, Dissertation, Universität Ulm, Medizinische Fakultät.
  3. Kurz A, Double KL, Lastres-Becker I, Tozzi A, Tantucci M, Bockhart V, Bonin M, García-Arencibia M, Nuber S, Schlaudraff F, Liss B, Fernández-Ruiz J, Gerlach M, Wüllner U, Lüddens H, Calabresi P, Auburger G and Gispert S (2010): A53T-alpha-Synuclein Overexpression Impairs Dopamine Signaling and Striatal Synaptic Plasticity in Old Mice. PLoS ONE 5(7): e11464. doi:10.1371/journal.pone.0011464http://www.ncbi.nlm.nih.gov/pubmed/20628651.
  4. Gründemann J, Schlaudraff F, and Liss B (2011): UV-laser microdissection and gene expression analysis of individual neurons from post mortem Parkinson's disease brains. Methods in Molecular Biology, bookchapter, in press.
  5. Begus-Nahrmann Y, Lechel A, Obenauf AC, Nalapareddy K, Peit E, Hoffmann E, Schlaudraff F, Liss B, Schirmacher P, Kestler H, Danenberg E, Barker N, Clevers H, Speicher MR, Rudolph KL: p53 deletion impairs clearance of chromosomal-instable stem cells in aging telomere-dysfunctional mice. Nature Genetics 41:10 (2009) 1138–43. Epub 2009 Aug 30
  6. Gründemann J, Schlaudraff F, Haeckel O and Liss B: Elevated alpha-synuclein mRNA levels in individual UV-laser-microdissected dopaminergic substantia nigra neurons in idiopathic Parkinson's disease. Nucleic Acids Research 36:7 (2008) e38. Epub 2008 Mar 10.
  7. Aguado C, Colon J, Ciruela F, Schlaudraff F, Cabanero MJ, Watanabe M, Liss B, Wickman K, Lujan R: Cell type specific subunit composition of G-protein-gated potassium channels in the cerebellum. J Neurochemistry 105:2 (2008) 497–511. Epub 2007 Dec 6.

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