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Isolation of Single Immunohistochemically Identified Whole Neuronal Cell Bodies from Post-Mortem Human Brain for Simultaneous Anlaysis of Multiple Gene Expression
Peter T. Nelson led this live discussion on 27 February 1998. Readers are invited to submit additional comments by using our Comments form at the bottom of the page. Paper Under Discussion: Cheetham JE, Coleman PD, Chow N. Isolation of Single Immunohistochemically Identified Whole Neuronal Cell Bodies from Post-Mortem Human Brain for Simultaneous Anlaysis of Multiple Gene Expression. Journal of Neuroscience Methods 77 (1997): 43-48. Abstract  View Transcript of Live Discussion — Posted 7 September 2006
Background Text
By Peter T. Nelson
Cheetham and colleagues describe a novel
and ingenious way of isolating brain cells and assaying for multiple RNA
transcripts, in which context one wishes to elucidate information about
the RNA in a given cell in comparison to the RNA of the cell's neighbors.
This technique has special application to neurodegenerative disease. The
following discussion includes my thoughts about the methods employed; figure-by-figure
analysis of results; and some discussion on the paper and some of the uses
of this new technology.
Methods
This is primarily a methods paper. The
method for isolating and analyzing a single cell's RNA is indicated on the
flow chart of Figure 1. The procedure is straightforward, appealingly so.
This is also, of course, a preliminary paper, and the authors will in follow-up
studies describe the application of the technique in more rigorous detail.
At the present stage, however, I am left with some questions:
Question 1) After a postmortem interval of up to 16 hrs, the brain is dissected;
1mm blocks are minced out; cells are physically smeared onto an adhesive
surface after trypsin treatment; immunocytochemistry is performed in which
huge antibody proteins have access to the cell cytoplasm; and the cells
are plucked away with a pipet. Is it not possible, after all of this dramatic
manipulation, that RNA could flow out of the one cell and into another,
rendering future PCR inaccurate?
Response by Nienwen Chow: During tissue processing, RNA from broken cells would be
degraded. That is why acridine orange staining on tissue smear (Figure
2A) does not show a general orange background. After fixation, we
believe that RNA are retained within the cells. If RNA leaking from
fixed cells is a problem, in situ hybridization and in situ RT-PCR would
not be possible.
Question 2) What effect of postmortem interval was noted?
Response by Nienwen Chow: The maximum postmortem interval for our tissues is 16 hours
(average is 6 hrs.), and all of the brains we have taken worked.
However, it only shows the presence of RNA from tissues with various
postmortem delay, but nothing about the quality of RNA. We never
studied the effect of postmortem delay on individual RNA species. For
information on that, please see the paper by Lukiw et al., (Int. J.
Neurosci., 1990, 55:81-88.).
Question 3) What percentage of the brains that were analyzed produced viable RNA
(only one was shown)?
Response by Nienwen Chow: 100 percent.
Question 4) How quantitative are these particular dot-blot hybridizations?
Response by Nienwen Chow: If radioactive probes are used and the hybridization
intensities are detected by Phosphimager, within a wide range of aRNA
abundance, the hybridization signal is linearly related to the
concentation of aRNA. In a recently submitted manuscript, we extended
the same study shown in Figure to 30 different cDNAs and showed the same
results.
Question 5) It would have been interesting to have included a different
cell population, e.g. GFAP- immunoreactive astrocytes.
Doing so would have demonstrated the flexibility of
the technique and allowed a comparison of the two cell
populations' RNA.
Results
Figure 2. Acridine orange and NF-H IHC reveal that on the (separate)
slide smears are cells that contain RNA and are positively stained for NF-H.
One is given the impression that these trypsin-treated cells are very close
to one another, and that as they were smeared on the slide they rolled over
onto one another. If a (very large) antibody protein could penetrate the
cell membrane, couldn't RNAs have transferred too? And if one elected to
pluck with a pipet one of the neurons in Figure 2B, might one be in danger
of having contamination from the other two nearby neurons? That being said,
it is impressive and attractive to have such isolated and characterized
cells as are being demonstrated here.
Response by Nienwen Chow: Again, there is a concern about RNA leakage. We have addressed
that in Methods 1).
Figure 3. A northern blot of aRNA transcripts from nine cells
of one human brain. Some of the transcripts are quite large. One wonders
if all the brains had RNA this good...
Response by Nienwen Chow: Yes.
Figure 4. Dot-blot hybridization of aRNA from a single cell using
aRNA of different concentrations. This is the crux of the paper. Here they
have cDNA clones blotted to nylon membranes being washed in a medium of
radiolabelled aRNA from isolated neurons. There are present numerous transcripts
in the aRNA; furthermore, the blots demonstrate linear increase in staining
intensity for the individual transcripts. Why such a low level of expression?
Is the number of actin transcripts in a nerve cell so low in comparison
to, say, cyclin D1?
Response by Nienwen Chow: We want to make a correction here. The actin cDNA used in the
study is actually an actin-bundling protein clone. A mistake was made by
the commercial vender of this cDNA clone. However, it remains a fact
that the hybridization intensities for some RNA transcripts with medium
abundance are very low. We noted that the intensity has something to do
with the sequence and the length of the cDNA used. The efficiency of
amplification may also be different for different transcripts, depending
on the length of the poly A tail. Other factors may also come into play
here.
Table 1. The result of "RNA profiling" from nine neurons
of a single control brain with five different cDNA dot-blot reverse probes.
It seemed from the table that there were some neurons that had relatively
higher values (e.g. cell #'s 1,6,9) and some had relatively low values (e.g.
cell #'s 2,3,8), and the basic proportions of one transcript to the other
was relatively constant (a1-ACT>GAPDH>cyclin D1=nestin). Is this finding
consistent with previously described quantities of these transcripts in
nerve cells?
Response by Nienwen Chow: In order to compare
data across sasmples, one needs to normalize the values
to an internal control. In this case, we did not think
any cDNA could serve as a good internal control. In
a recently submitted manuscript, we proposed to use
the average of all measurements (cDNAs) as an internal
control.
Cheetham and colleagues here introduce
a new way of analyzing RNA from individual characterized neurons isolated
from the human brain. The strength of the experimental technique is, firstly,
that it appears to work sometimes (nothing to be taken for granted!). Secondly,
it offers the chance to compare "sick" cells to cells nearby that
don't appear afflicted. This has special application to diseases such as
Alzheimer's, Parkinson's, Pick's, progressive supranuclear palsy, Lewy Body
Disease, inclusion body myositis, and others, in which there are obvious
manifestations within "sick" cells (various inclusion bodies);
e.g. how does a tangle-bearing cell differ from a non-tangle-bearing cell
re: mRNA transcripts? Lastly, when you have transcribed aRNA in a test-tube
from a single cell, it allows for all sorts of manipulation (e.g. analysis
of splicing changes) for that given cell that other techniques, such as
in situ hybridization, cannot approach. This technique surely holds great
promise--that is the central message of the paper.
It seems premature to discuss this technique with a deeply critical eye,
because the present paper does not seem a deep demonstration of it. If the
article purports to demonstrate producing RNA from isolated characterized
neurons, it has yet to truly describe the quality of the RNA or fully dispel
concerns of contamination between neurons. It would be nice to see the RNA
produced from many cell types; from many brains; and from the brains of
patients with disease. It would be nice to see a more complex description
of the quality of RNA that was produced. It would be nice to have an explanation
of why only six of 53 neurons produced PCR- quality RNA.
Response by Nienwen Chow: We do not feel that the method needs to be limited to cells with
inclusion bodies as is implied by Dr. Nelson. Any characteristic that
can be detected by immunohistochemistry may be used to select cells.
Detection of cell properties of interest need not be limited to
immunohistochemistry, but could encompass any method that retains the
integrity of the RNA.
"It would be nice to see the RNA produced from many cell types . . ."
Response by Nienwen Chow: In our ms. recently submitted we limit ourselves to one cell type, but 5
brains representing different disease states.
"It would be nice to have an explanation of why only 6 of 53 neurons
produced PCR quality RNA"
Response by Nienwen Chow: This PCR was done with primers for G3PDH and now we use primers for 18s
ribosomal RNA and the rate of positives is roughly 50 percent or higher. This
seems to imply an issue of sensitivity of PCR rather than the quality of
the RNA.
A few pitfalls of the technique come to mind:
Comment:1) RNA degradation seems inevitable. Some messages will be degraded and
others won't; hence, any conclusion drawn using this technique may have
systematic errors.
Response by Nienwen Chow: True. Answered in part above. In addition, comparison of "diseased"
and "normal" cells from the same region of the same brain obviates this
issue to some extent.
Comment:2) Let us consider the example of studying the differences between "normal"
and "tangle-bearing" cells' RNAs. Let us say that a certain message
is lessened in the tangle-bearing cells. Perhaps this difference would only
be due to a difference in how the different cells react to postmortem artifact.
Even if tangle-bearing cells have an increase in a message, that too could
be due to some artifact of the technique. (Parenthetically, it should be
added that an animal model of neurofibrillary changes, such as the aged
sheep, could come in handy in this situation).
Response by Nienwen Chow: True. This problem also applies to in situ hybridization,
immunohistochemistry, etc. Use of tangle and non-tangle neurons from
aged sheep is a good suggestion, with the caveat that the mechanism
giving rise to NFT in sheep may differ from that producing NFT in humans
with AD.
Comment:3) Another critique of the paper is that in situ hybridization allows
for quantitation of messages in cells. Dr. Coleman and colleagues have been
fruitfully comparing messages from tangle- and non-tangle-bearing cells
for years.
That being said, the advantages of having individual cells' RNA for comparison
of pathological cells vs. normal cells, and of cells from pathological vs.
normal brains, are great, and the implications probably surpass the imagination
of this journal club reviewer. The work of Cheetham and colleagues is of
great interest to anyone in the field and one may anticipate work of great
significance being produced soon from that laboratory.
Response by Nienwen Chow: We are not sure in which way this is a critique. Studying one
message at a time with in situ hybridization demonstrated to us
dramatically that expression of some messages increases in NFT neurons,
and decreases for other messages. It seemed to us that characterizing
profiles could be accomplished more efficiently with the aRNA method
than with the one message at a time of in situ hybridization. We
continue to use both methods and, in fact, for selected messages confirm
results by both methods. For example, our aRNA data have suggested that
NF-M is unchanged in AD neurons. This was confirmed by in situ
hybridization study in our lab. We attribute the previously reported
decreases in NF-M expression demonstrated by Northern blots to loss of
neurons.
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