Báo cáo khoa học: RCAN1-1L is overexpressed in neurons of Alzheimer’s disease patients pot

It is also unknown how many protein isoforms are expressed in human brain and whether RCAN1 protein is overexpressed in Alzheimer’s disease.. Using western blotting, we now show that the

disease patients

Cathryn D Harris, Gennady Ermak and Kelvin J A Davies

Ethel Percy Andrus Gerontology Center, and Division of Molecular & Computational Biology, The University of Southern California,

Los Angeles, CA, USA

The RCAN1 gene is located on human chromosome 21

in region q22.12 (Fig 1) [1] Initially thought to lie

within the Down’s syndrome critical region, it was

sub-sequently found to lie outside of this region RCAN1

consists of seven exons, which can undergo alternative

splicing to produce different mRNA isoforms and,

con-sequently, different proteins (Fig 2) [2] A cluster of 15

putative nuclear factor of activated T-cells

(NFAT)-binding sites lie in the intron, just 5¢ to exon 4 [3] All

known mRNA isoforms contain exons 5–7, and the

three isoforms most studied also contain either 29 amino

acids (now RCAN1-1 ‘Short’ or RCAN1-1S), or 55 amino acids (RCAN1-1 ‘Long’ or RCAN1-1L) encoded

by exon 1, or 29 amino acids (RCAN1-4) encoded by exon 4 (Fig 2) It has been suggested that isoform 4 may be initiated by an alternative, calcineurin-respon-sive, promoter, due to the cluster of 15 NFAT-binding elements 5¢ to exon 5 [4] A splice variant containing exon 2 has been reported in fetal liver and brain [2], but

no isoforms containing exon 3 have yet been described The RCAN1 protein is able to bind to and inhibit the catalytic subunit of calcineurin (protein phosphatase 2B)

Keywords

Alzheimer’s disease; calcipressin 1; DSCR1;

Adapt78; RCAN1

Correspondence

K J A Davies, Ethel Percy Andrus

Gerontology Center, University of Southern

California, 3715 McClintock Avenue,

Los Angeles, CA 90089-0191, USA

Fax: +1 213 740 6462

Tel: +1 213 740 8959

E-mail: kelvin@usc.edu

Note

The new name RCAN1 (regulator of

cal-cineurin) has recently been accepted by the

HUGO Gene Nomenclature Committee for

the gene previously known as DSCR1 or

Adapt78 Similarly, RCAN1 is the new name

for its protein product, which was previously

know as calcipressin 1 or MCIP1

(Received 24 April 2006, revised 16

Decem-ber 2006, accepted 29 January 2007)

doi:10.1111/j.1742-4658.2007.05717.x

At least two different isoforms of RCAN1 mRNA are expressed in neuro-nal cells in normal human brain Although RCAN1 mRNA is elevated in brain regions affected by Alzheimer’s disease, it is not known whether the disease affects neuronal RCAN1, or if other cell types (e.g astrocytes or microglia) are affected It is also unknown how many protein isoforms are expressed in human brain and whether RCAN1 protein is overexpressed in Alzheimer’s disease We explored the expression of both RCAN1-1 and RCAN1-4mRNA isoforms in various cell types in normal and Alzheimer’s disease postmortem samples, using the combined technique of immunohist-ochemistry and in situ hybridization We found that both exon 1 and exon 4 are predominantly expressed in neuronal cells, and no significant expression of either of the exons was observed in astocytes or microglial cells This was true in both normal and Alzheimer’s disease brain sections

We also demonstrate that RCAN1-1 mRNA levels are approximately two-fold higher in neurons from Alzheimer’s disease patients versus non-Alzhei-mer’s disease controls Using western blotting, we now show that there are three RCAN1 protein isoforms expressed in human brain: RCAN1-1L, RCAN1-1S, and RCAN1-4 We have determined that RCAN1-1L is expressed at twice the level of RCAN1-4, and that there is very minor expression of RCAN1-1S We also found that the RCAN1-1L protein is overexpressed in Alzheimer’s disease patients, whereas RCAN1-4 is not From these results, we conclude that RCAN1-1 may play a role in Alzhei-mer’s disease, whereas RCAN1-4 may serve another purpose

Abbreviations

AD, Alzheimer’s disease; Cb, cerebellum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase gene; GFAP, glial fibrillary acidic protein; Hc, hippocampus; HLA-DR, human leukocyte antigen-DR; LA, long and accurate; NeuN, neuronal nuclei; NFAT, nuclear factor of activated T-cells.

[3,5] Calcineurin is a calcium-dependent serine–

threonine protein phosphatase, which has several

known substrates, including the transcription factor

NFAT, which is well characterized, and the tau

pro-tein We have proposed that RCAN1 may have a role

in the development of Alzheimer’s disease (AD) (and

other ‘tauopathies’), because it inhibits calcineurin

from dephosphorylating the tau protein, resulting in

hyperphosphorylated tau, which may then promote the

formation of paired helical filaments and

neurofibril-lary tangles [6–8] RCAN1 is chronically overexpressed

in AD, presumably due to to the stress of chronic inflammation [6–8] There are data showing decreased calcineurin activity in AD, and other studies have shown that calcineurin inhibition results in tau phos-phorylation on serine and threonine residues, consis-tent with those that occur in AD [9–13] RCAN1 is expressed primarily in neurons in both rat and human brain tissue [6] Importantly, this complements data from rat tissues showing that calcineurin is also expressed in neurons [14,15]

RCAN1 gene expression is significant in several tis-sues, particularly human brain, spinal cord, kidney, liver, mammary gland, placenta, skeletal muscle, and heart [6] We have previously found that there are

Detection of RCAN1 Isoforms with Various Antibodies

Quantification of RCAN1-1L and RCAN1-4 Isoforms

RCAN1 Antibody Used

Isoform Detected

RCAN1 Expression in Human Brain A

B

Fig 2 RCAN1 protein expression in human brain Using an anti-body directed at exon 7, which should recognize all RCAN1 iso-forms, three bands were detected by western blotting (A) These bands appear at 70, 38 and 31 kDa All three bands were present regardless of the brain region tested or the presence or absence of disease, although the 31 kDa band, in some cases, was very faint Using antibodies specific to exon 1 or exon 4, we have identified the 70 kDa band as RCAN1-4, the 38 kDa band as RCAN1-1L, and the 31 kDa band as RCAN1-1S Combined data from western blots from 12 control and 12 AD patients, in all regions tested (A10, A22,

Hc and Cb), were quantified by densitometry, and standard errors were calculated (B) Fisher’s test was performed to analyze whe-ther differences were statistically significant In these samples, RCAN1-1L was expressed at a level approximately two-fold higher than RCAN1-4 (P < 0.05), a significant difference.

RCAN1 Structure

4 5 6 7

1 5 6 7

1 5 6 7

FLISPP

RCAN1-1S Protein

197 Amino Acids 29

CaN binding motif (PKIIQT)

197 Amino Acids 29

252 Amino Acids 55

Chromosome 21

3 2 2.

15 NFAT

binding sites

RCAN1 Genomic DNA

RCAN1-1L Protein

RCAN1-4 Protein

DSCR

Fig 1 Structure of the RCAN1 gene and the RCAN1 protein

Chro-mosome 21 ) Human RCAN1 is located on chromosome 21 in

region q22.12, just outside of the Down’s syndrome candidate

region RCAN1 genomic DNA ) RCAN1 consists of seven exons

that are alternatively spliced and vary in their 5¢-exon, but all contain

exons 5, 6, and 7 There is a cluster of 15 NFAT-binding sites on

the RCAN1 gene, 5¢ to exon 4 which may function as an alternative

promoter region for the exon 4 splice variant RCAN1 protein ) We

have found evidence for three RCAN1 protein isoforms in human

brain, RCAN1-1S, RCAN1-1L, and RCAN1-4 (see Fig 2 for these

data) All RCAN1 isoforms differ in their initial exon, but share the

168 amino acids encoded by exons 5, 6 and 7, as well as the

con-served FLISPP sequence found in all of the RCAN1 family

mem-bers This motif shares homology with the serine–proline (SP)

boxes found in NFAT protein family members All RCAN1 isoforms

contain a putative calcineurin-binding motif (PKIIQT).

at least two isoforms of RCAN1 expressed at

signifi-cant levels in human brain (RCAN1-1 and RCAN1-4),

and that, in general, RCAN1 is overexpressed in AD

only in regions actually affected by the disease [6]

RCAN1 has also been shown to be upregulated in

Down’s syndrome postmortem brain tissue [5,6], and it

is interesting to note that Down’s syndrome patients

also suffer from an early-onset form of AD It is

poss-ible that RCAN1 may be protective when expressed

transiently, but may be part of a maladaptive response

if its expression fails to be turned off, resulting in

dis-ease conditions

There is, as yet, no explanation for why cells have

multiple isoforms of this gene and protein, or what the

differences in function of each form of the gene and

protein may be We hypothesized that there might be

differences either in the levels of RCAN1 isoform

expression, or in the cellular localization of expression,

in brain regions affected by AD We therefore felt that

it was first important to test for the expression of

dif-ferent RCAN1 mRNAs and proteins in AD human

brain tissue as compared to that of age-matched

controls Second, we felt that it was important to

investi-gate the cellular distribution of the isoforms in

brain-specific cell types: neurons, microglia, and astrocytes

Results

RCAN1 isoform expression in human brain

Previous work from our laboratory has shown that

RCAN1 mRNA is significantly expressed in adult

human brain, and upregulated in those brain areas

affected by AD Although both isoforms 1 and 4 of

the RCAN1 gene are expressed in brain tissue, no one

has reported any differences in function or localization

of these isoforms in the brain In order to try to

understand how these isoforms may differ, we

exam-ined the expression of the isoforms known to be

tran-scribed in brain tissue, isoforms RCAN1-1 and

RCAN1-4 To determine which, if any, protein

iso-forms were expressed, brain tissue extracts were

pre-pared for western blotting These blots were first

probed with an antibody raised against exon 7, which

is a portion of the C-terminus of RCAN1 This region

is common to all predicted isoforms, and the antibody

should therefore recognize all forms of the RCAN1

protein The antibodies were first tested on cell extracts

to ensure reactivity After the antibody had been

affin-ity purified, it recognized two major bands, and one

very light band, in brain lysates by western blot

analy-sis (Fig 2A) These bands resolved at approximately

70, 38 and 31 kDa on denaturing polyacrylamide gels

Next, antibodies specific for exon 1 or exon 4 were tested on adult human brain tissues, again using west-ern blotting, to try to match each band with the unique isoform of the RCAN1 protein to which it cor-responded The band around 70 kDa was recognized

by the exon 4 antibody as RCAN1-4, and the 38 and

31 kDa bands were recognized by the exon 1 antibody

as RCAN1-1L and RCAN1-1S isoforms, respectively (Fig 2A) As this antibody is generated against a pep-tide present in both RCAN1-1 isoforms, it recognizes both bands RCAN1-1S was the minor band, which was very weak and difficult to detect and quantify The densities of the RCAN1-4 and RCAN1-1L bands recognized by the common antibody were quanti-fied using ipgel software (Scanalytics, Vienna, VA) (Fig 2B) In good agreement with our previous work on RCAN1 mRNA isoforms in brain [6], the RCAN1-1L protein was expressed at a much higher level than was the RCAN1-4 protein The RCAN1-1L protein concen-tration was approximately double that of RCAN1-4 in whole brain homogenates (combined regions) However, our antibody specific for exon 4 binds with much greater affinity to the RCAN1-4 protein, and produces a pro-portionately stronger signal, than does our RCAN1-1 antibody (specific for exon 1), even though there is a greater amount of RCAN1-1L Thus, the actual quanti-ties of RCAN1-1 and RCAN1-4 can only be directly compared in western blots using the common antibody, containing the exon 4 sequence

RCAN1-1L is overexpressed in AD Northern blots show that RCAN1 mRNA is

upregulat-ed in regions of the brain that are affectupregulat-ed by AD, as well as in a non-AD patients with neurofibrillary tan-gles [6] In this study, protein extracts originate from regions of the brain including the cerebellum (Cb), which should not be affected by AD and therefore can serve as an internal control, and regions that are affec-ted by AD, including the cerebral cortex (regions A10 and A22) and the hippocampus (Hc) To ensure that effects were due to actual differences, and not loading, membranes were stained with Ponceau S, and all sam-ples were normalized to loading controls We found that RCAN1-1L was upregulated in brain regions affected by AD as compared to control tissues (a rep-resentative western blot is shown in Fig 3A)

As human brain tissue is difficult to obtain, we focused on the most interesting regions for further studies These regions included the Hc and the Cb (for internal control) We found that there was significant upregulation of RCAN1-1L in the Hc of AD patients, but regulation did not appear to be significant for

RCAN1-4 (Fig 3B) Using Fisher’s protected least

significant difference (PLSD) test on RCAN1-1L

expression data, AD Hc was significantly different

from control Hc (P < 0.05) Using Fisher’s PLSD test

on RCAN1-4 expression data, no group was

signifi-cantly different from any other As the RCAN1-1S

isoform was difficult to detect, and represents only a

very minor fraction of RCAN1-1 expression, we have

not included it here

Cellular distribution of RCAN1-1 mRNA in human

brain

As RCAN1-1 protein was upregulated in AD tissues,

we wanted to determine if there were any differences

in which brain cell types expressed RCAN1 We again

focused on RCAN1-1, as it was upregulated in AD, using the combined techniques of in situ hybridization and immunocytochemistry In this experiment, expres-sion of RCAN1-1 was identified using an antisense RNA probe against exon 1 Expression was examined

in neurons, astrocytes and microglia, by labeling cells with antibodies against each of these specific cell types

We first created a construct that could produce both

an RCAN1-1 antisense and sense (control) transcript for use as a radiolabeled probe (Fig 4A) Our anti-sense probe hybridized to tissue sample, as shown by clusters of black grains, whereas our control, sense, probe did not hybridize and only showed scattered background grains (Fig 4B) This indicates that our system was working correctly

Next we tested samples by labeling neurons, astro-cytes, or microglia We found that in both control and

AD postmortem samples, expression of RCAN1-1, as shown by clusters of grains, highly colocalized with neuronal cells and not with astrocytes or microglia (Fig 4C) The clusters were also larger and denser in

AD samples as compared with control samples This is

in good agreement with our previously reported nor-thern blot data, showing that RCAN1 mRNA expres-sion is greater in AD than in age-matched control samples [6] Expression of RCAN1-4 also localized to neurons, although, as it is expressed at low levels, its concentration was still not dramatically higher than background levels (Fig 4C)

RCAN1-1 mRNA is overexpressed in neuronal cells of AD patients

We examined mRNA expression of RCAN1 in brain tissue from AD and age-matched control samples by RT-PCR of cDNA (Fig 5A) Upon quantification of PCR, our results showed a clear upregulation of RCAN1-1 mRNA in the primary region that is affected

by AD, the Hc (Fisher’s P-value of < 0.05) Expression was not significantly increased in the Cb, as would be expected, as this region is not affected by AD (Fig 5B) When quantifying mRNA expression in neurons from our combined in situ hybridization–immunocyto-chemistry, we obtained similar results to the RT-PCR data above By quantifying the grain cluster density associated with a neuron, and subtracting the back-ground expression density, expression of mRNA in

AD and control samples can be determined With this method, it appears that expression of RCAN1 is almost doubled in AD (Fisher’s P-value of < 0.05) compared to control samples (Fig 5C)

The increase in RCAN1-1 mRNA levels seen in the

Hc (but not the Cb) of AD patients in Fig 5B,C is in

Fig 3 RCAN1-1 is overexpressed in AD RCAN1-1L and RCAN1-4

protein expression was measured via western blot [a

representa-tive blot is shown in (A)] in controls and AD patients Blots

contain-ing control and AD patient samples were probed with antibody

against exon 1, stripped, and then probed with antibody against

exon 4 Ponceau S staining of membranes, and probing of blots

with b-tubulin antibody, were used to control loading levels In (B),

densities of the bands from 12 control and 12 AD patient samples

were quantified using IPGEL Laboratory software, and normalized to

a b-tubulin loading control, and standard errors were calculated.

Fisher’s test was performed to analyze whether differences were

statistically significant The only significant difference (producing a

P-value of < 0.05) between the control and AD samples found was

in the RCAN1-1 protein in the Hc (marked with an asterisk) As

RCAN1-1 protein expression was approximately double that of

RCAN1-4 (Fig 2B), the signal strength of the two isoforms has

been adjusted accordingly in this figure.

good agreement with the increase in hippocampal

RCAN1-1 protein levels reported for AD patients in

Fig 3C Thus, it is possible that elevated RCAN1-1

protein concentrations in AD are the result of

tran-scriptional upregulation; this possibility will now have

to be rigorously tested

Discussion

RCAN1 has been shown to bind to and inhibit the

ser-ine–threonine protein phosphatase calcineurin [5] The

brain is an especially interesting organ in which to

examine RCAN1 expression, because calcineurin is

highly expressed in this organ, comprising

approxi-mately 1% of total protein We have hypothesized that

a role for RCAN1 in the development of neurodegen-erative ‘tauopathies’, such as AD, is that it may inhibit calcineurin from dephosphorylating the tau protein, resulting in hyperphosphorylated tau, which may then promote the formation of paired helical filaments and neurofibrillary tangles [6–8] This fits nicely with data from other studies showing decreased calcineurin activ-ity in AD, and other data showing that calcineurin inhibition results in tau phosphorylation on serine and threonine residues consistent with those that occur in

AD [9–13]

In the studies presented in this article, we provide evi-dence for the presence of at least three distinct RCAN1

C

Fig 4 Analysis of RCAN1 mRNA

expres-sion in human brain Probes for in situ

hybridization were created by cloning

RCAN1-1 into the multiple cloning site of

the pBluescript II SK(+ ⁄ –) vector (A) Use of

this vector allowed for both sense (control)

and antisense probes to be produced from a

single clone The sense probe did not

hybridize to the sample, whereas the

anti-sense probe did (B) In all slides, specific

cell types (either neurons, astrocytes or

microglia) are immunochemically stained

with diaminobenzidine and appear brown.

Cell type-specific antibodies used were:

anti-NeuN mAb for neurons, anti-GFAP for

astrocytes, and anti-HLA-DR for microglia,

and are shown at a magnification of 200·.

Expression of RCAN1-1 mRNA was

detec-ted by in situ hybridization, in which

hybrid-ization produces clusters of black grains.

Representative samples show that clusters

align with neurons in both control and AD

samples, but not with astrocytes or

micro-glia (C) Expression is clearly higher in AD

neurons, because these clusters are denser.

protein isoforms in human brain (Fig 2A) We now

demonstrate that two of the possible protein isoforms,

RCAN1-1L and RCAN1-4, appear to be highly

expressed in brain, whereas RCAN1-1S is expressed at

very low levels (Fig 2A) Our antibodies detect

RCAN1-4 at approximately 70 kDa, which is about

twice as large as the RCAN1-1S protein This is also

much larger that has been described in other tissues

(25–29 kDa) There are several possible explanations

for this First, there are additional stop codons located

in exon 7 One of these would produce a peptide

con-taining 595 amino acids, which would produce a protein

with a predicted size of 67 kDa, and another would

pro-duce a peptide containing 632 amino acids, which

would have a predicted size of 71.7 kDa Another

explanation is that the protein may form a covalent

dimer (not a disulfide-linked dimer) that is not

separ-ated by SDS⁄ PAGE The expression of RCAN1-1L

protein was approximately double that of RCAN1-4 in

general, as determined by quantifying the densities of

bands detected with a common antibody that recognizes all isoforms of the RCAN1 protein, in all regions of the brain, and in samples from both AD and control patients (Fig 2B) Northern blots show that RCAN1 expression is upregulated in regions of the brain affec-ted by AD, as well as in a non-AD patient exhibiting neurofibrillary tangles [6] Therefore, RCAN1-1 may be related to this particular AD pathology

Expression of the RCAN1-1L protein was greater in

AD patients as compared to age-matched control patients We found that, regardless of the isoform, RCAN1 was expressed in each region of brain tested,

in both AD and control samples (Fig 3A) We found, however, that RCAN1-1L was the only isoform clearly upregulated in AD, as compared to age-matched con-trol samples (Fig 3B) Thus, RCAN1-1L may play a role in AD, whereas RCAN1-4 does not appear to be involved in this pathology

As RCAN1-1 protein is overexpressed in AD, we next examined its mRNA transcript expression at the cellular level, to see if there were any differences in localization between AD and control samples As RCAN1-1S represents a minor proportion of total

A

B

C

Fig 5 RCAN1 mRNA is overexpressed in AD (A) RCAN1-1 mRNA expression was detected using RT-PCR in AD and control samples Amplification of GAPDH was used as a loading control.

A10–A10, cerebral cortex area; A22–A22, cerebral cortex area RNA was amplified using LA RT-PCR for 30 cycles: 98 C for 20 s, followed by 68 C for 3 min (B) The amount of input cDNA in each sample was equalized by amplification of the GAPDH gene.

To ensure that GAPDH amplification was quantitative, we ran serially diluted cDNA samples for different numbers of cycles Typically, it took about 25 cycles to achieve a linear dependency between the amount of input DNA and the resulting PCR prod-ucts Then, equal amounts of the cDNA (according to amplifica-tion of control GAPDH fragment) were used to estimate the amount of RCAN1-1 mRNA As with GAPDH amplification, seri-ally diluted cDNA samples were run for different numbers of cycles to find conditions in which the amount of amplified RCAN1-1 fragments was proportional to the amount of the input cDNA in the reactions (C) Radioative In situ hybridization was performed to label either RCAN1-1 or RCAN1-4 expression This technique was combined with immunocytochemistry to label spe-cific cell types with antibodies Anti-NeuN mAb was used to label neurons, GFAP was used to label astrocytes, and anti-HLA-DR was used to label microglia In this experiment, each slide contained a set of one AD patient and one control patient section, in triplicate The hybridization signal of RCAN1-1 was quantified in neurons by counting grain density on neurons and subtracting background grain density levels Standard errors were calculated, and Fischer’s test was performed to analyze signifi-cance This shows approximately a two-fold increase in RCAN1-1 mRNA in the four Alzheimer’s disease versus four control patient tissue samples.

RCAN1 expression, we reasoned that signals detected

by our probe against exon 1 would be predominantly

due to expression of RCAN1-1L We found that

RCAN1-1 is expressed in neurons in both AD and

control samples as detected by in situ hybridization

(Fig 4C) It does not appear to be expressed in

micro-glia or astrocytes RCAN1-4 is expressed at a much

lower level, and therefore difficult to detect by this

method, but also appears to be expressed in neurons

When RCAN1-1 expression is measured by RT-PCR,

it is also seen to be upregulated selectively in brain

regions affected by AD, as compared to (age-matched)

control patients (Fig 5A) Quantification of RCAN1-1

mRNA expression in neurons also shows that it is

upregulated in AD (Fig 5B)

Both RCAN1-1 and RCAN1-4 are expressed in

brain tissue in both control and AD patients

RCAN1-4 is expressed at a much lower level,

how-ever, and it does not appear to play a role in this

disease RCAN1-4 is under the control of an

alter-native promoter and is also feedback regulated,

which may account for, at least in part, differences

in regulation of the different RCAN1 isoforms [4] It

has been shown that RCAN1-4 is expressed as a

stress-protective protein [16], which can arrest cell

growth, whereas RCAN1-1 can induce cellular

growth [7,17] The data presented in this article show

that RCAN1-1 is upregulated at both the mRNA

and protein levels in AD, and therefore may

contrib-ute to disease pathology RCAN1-1 appears to be

preferentially expressed in neurons, rather than

astro-cytes or microglia, in both normal brain tissue and

brain samples from AD patients Therefore, there

are differences in levels of RCAN1-1 expression, but

there do not appear to be differences in the cell type

in which the different isoforms are expressed

RCAN1-1 is upregulated not only in AD, but also

in non-AD brain tissue that exhibits one of the AD

hallmarks: neurofibrillary tangles Chronically

eleva-ted RCAN1-1 levels may, thus, cause an increase in

phosphorylation of the tau protein, leading to the

formation of neurofibrillary tangles in a variety of

neurodegenerative tauopathies

Experimental procedures

Postmortem human brain tissue

The brain samples used in this project were graciously

pro-vided by the Alzheimer’s Disease Research Center at the

University of Southern California’s Keck School of

Medi-cine, Los Angeles, CA Brain tissues, with a postmortem

interval of less than 6 h, were fresh frozen at) 70 C until

use Samples analyzed in this study originated from the Hc, cerebral cortex region A10, cerebral cortex region A22, and the Cb All samples were accompanied by Alzheimer’s Dis-ease Research Center neuropathology summaries and AD samples, and all displayed between moderate and severe disease pathology

Antibodies

Antibodies to exon 7 (the common C-terminal region), exon 1 and exon 4 of the RCAN1 gene were custom pro-duced against peptides injected into rabbits, and affinity purified by ProSci Incorporated (Poway, CA) An exon 1 antibody was generated against the peptide NH2 -MEEVDLQDLPSAT-OH, and an exon 4 antibody was produced against the peptide NH2

-VANSDIFSESETR-OH The antibody against exon 7 was created as previ-ously described [7] After production, sera, purified anti-bodies and flow-through were tested, along with competitive binding assays Commercially produced b-tub-ulin and secondary antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA) Experimental animals were handled according to NIH guidelines for the care and use of laboratory animals

Western blotting

Extracts were prepared by homogenization in cell lysis buffer (1· NaCl ⁄ Pi, 1% Igepal, 0.1% SDS, 0.1 mgÆmL)1 phenylmethanesulfonyl fluoride, 1 lgÆmL)1 leupeptin,

1 lgÆmL)1pepstatin A, 1 lgÆmL)1antipain, 10 lgÆmL)1 soy-bean trypsin inhibitor) and were cleared by centrifugation

at 16 000 g after incubation on ice for 30 min Protein con-centrations were determined using the BCA protein assay kit (Pierce, Rockford, IL), and equal amounts (20 lg of each sample) were loaded onto SDS polyacrylamide gels for fractionation The samples were electrophoretically transferred onto poly(vinylidene difluoride) membranes and stained with Ponceau S to verify loading The membranes were then blocked in 5% nonfat dry milk (Bio-Rad, Hercu-les, CA) with 0.1% Tween-20, and washed three times in wash solution (NaCl⁄ Pi with 0.1% Tween-20) The mem-branes were then probed with primary antibody at a dilu-tion of 1 : 1000, washed in washing soludilu-tion three times, and then probed with a horseradish peroxidase-conjugated secondary antibody at a dilution of 1 : 10 000 (Santa Cruz Biotech) Membranes were washed three more times in wash solution, and then visualized by use of the enhanced chemiluminescent reagent (ECL kit; Amersham, Piscata-way, NJ) and autoradiograpy Films were scanned, and expression was quantified using ipgel software Bands were normalized to b-tubulin expression Membranes were stripped in Pierce strip buffer and reprobed Statistical ana-lysis of western blot data was performed using statview software, using Fisher’s PLSD test for significance

RNA isolation

Total RNA was extracted using the TRIzol reagent (Life

Technologies, Gaithersburg, MD) The RNA concentration

was quantified spectrophotometrically, and relative content

was further confirmed with ethidium bromide-stained

gels Integrity of the RNA was estimated by agarose gel

electrophoresis Only RNA samples displaying discrete 28S

and 18S bands were used in experiments

Northern hybridization

Samples containing 10 lg of total RNA were subjected to

electrophoresis through 1% agarose formaldehyde gels,

blotted onto nylon membranes (Oncor, Gaithersburg, MD)

with HETS (CINNA⁄ BIOTECX, Houston, TX), and

cross-linked by ultraviolet radiation The membranes were then

prehybridized for 4 h and hybridized for 15 h in Hybrizol I

(Oncor) at 42C They were washed with 2 · NaCl ⁄

Cit + 0.1% SDS at room temperature for 1 and 10 min,

and then with 0.1· NaCl ⁄ Cit + 0.1% SDS at 60 C for 10

and 30 min The membranes were exposed, developed, and

scanned using the PhosphoImager system (Molecular

Dynamics, Sunnyvale, CA) To rehybridize blots, probes

were removed by washing membranes in a solution

contain-ing 0.1· NaCl ⁄ Cit + 0.1% SDS and 10 mm Tris ⁄ HCL

(pH 7.0) at 90C for 10 min To quantify levels of RCAN1

mRNA, the membranes were scanned, and the

hybridiza-tion signal was measured using imagequant software

(Molecular Dynamics) Each signal was recalculated

according to the amount of RNA actually loaded onto

the gels The amount of the loaded RNA was controlled

using a glyceraldehyde-3-phosphate dehydrogenase gene

(GAPDH) probe Probes containing [32P]dCTP[aP]-labeled

DNA were prepared using the High Prime system

(Boehrin-ger Mannheim, Mannheim, Germany) A PCR fragment

corresponding to RCAN1 isoform 1 was used to prepare

the RCAN1 probe, and a PCR fragment consisting of

GAPDH exons 7 and 8 was used to prepare GAPDH

probes

In situ hybridization

Brain samples were sectioned and mounted onto positively

charged slides Each slide contained samples from one

spe-cific brain region, with alternating AD and control samples

Immediately prior to use, sections were air-dried and fixed

in freshly prepared 4% buffered paraformaldehyde The

samples were then treated in acetic anhydride with 0.1 m

triethanolamine, and then rinsed and dehydrated in an

ethanol series and dried Slides were incubated in

prehy-bridization solution [50% formamide, 0.75 m sodium

chlor-ide, 0.05 m sodium phosphate buffer (PB, pH 7.4), 0.01 m

EDTA, 0.15 mm dithiothreitol, 1% SDS, 5· Denhardt’s

solution, 0.2 mgÆmL)1 heparin, 0.5 mgÆmL)1 tRNA,

0.05 mgÆmL)1polyA and polyC, and 0.25 mgÆmL)1sheared salmon sperm DNA] for 30 min at 53C in humidified chambers Prehybridization solution was then removed, and slides were hybridized to either antisense or sense (control)

35

S-labeled probes, cover-slipped, and incubated at 53C for 3 h in hybridization solution (prehybridization solution plus 10% dextran sulfate)

Slides were soaked in 4· NaCl ⁄ Cit and 100 mm b-merca-ptoethanol to remove coverslips After coverslips were removed, and slides were soaked in 0.5 m sodium chloride and 0.05 m phosphate buffer pH 7.4 for 10 min at room temperature; this was followed by incubation with 0.025 mgÆmL)1 RNaseA in 0.5 m sodium chloride and 0.05 m PB, for 30 min at 37C The slides were then washed

in a criterion wash solution, containing 50% formamide, 0.5 m sodium chloride, 0.05 m PB and 100 mm b-mercapto-ethanol, for 30 min at 50C, and then finally washed over-night in 0.5· NaCl ⁄ Cit and 20 mm b-mercaptoethanol

RNA probe preparation

Exon 1 and exon 4 sequences of RCAN1 were amplified from human cDNA by RT-PCR, using the LA-PCR kit (TaKaRa Bio Inc., Kusatsu, Japan) Primers used to amplify exon 1 consisted of the first 25 bases of exon 1 (5¢-GACTGGAGCTTCATTGACTGCGAGA-3¢) and the last 24 bases of exon 1 (5¢-CCGGCACAGGCCGTCCACG AACAC-3¢); primers for amplifying exon 4 consisted of the first 25 bases of exon 4 and the last 25 bases of exon 4 (5¢-CCTGGTTTCACTTTCGCTGAAGATA-3¢) Amplified fragments were then sequenced, and correct sequences were cloned into the SmaI site of the pBluescript II SK vector, between the recognition sites for the T3 and T7

polymeras-es, so that both antisense and sense (control) RNA probes could be produced from the same plasmid To verify that the correct sequence was inserted, and to determine the orientation of the insert, all clones were sequenced

These plasmids were transfected into Epicurian Coli XL2-Blue ultracompetent cells (Stratagene, La Jolla, CA), and grown Plasmids were collected using the Wizard Plus Miniprep kit (Promega, Madison, WI), and digested with the appropriate restriction enzyme Digestion of the template was confirmed by resolution on an agarose gel Probes were produced using the Riboprobe in vitro Transcription System (Promega), labeled with 35S accord-ing to the manufacturer’s protocol, and purified usaccord-ing Mini Quick Spin columns (Qiagen, Valencia, CA) Probes were then precipitated and dissolved in hybridization solution

Immunocytochemistry

Immediately following in situ hybridization, samples were rinsed twice in NaCl⁄ Pi, and endogenous peroxidases were blocked in NaCl⁄ Pi containing 10% methanol and 0.3%

hydrogen peroxide After being washed in NaCl⁄ Pi, slides

were treated with 1% NP-40 in NaCl⁄ Pi, and then washed

again in NaCl⁄ Pi After blocking for 30 min in blocking

solution (NaCl⁄ Pi, 0.01 mgÆmL)1heparin, 10 lm

dithiothre-itol, 100 unitsÆmL)1RNase inhibitor, and 3 lLÆmL)1sera),

samples were incubated with primary antibody for 90 min

Cell type-specific antibodies used were: anti-neuronal nuclei

(NeuN) IgG from Chemicon (Temecula, CA) for neurons

(1 : 500), anti-(glial fibrillary acidic protein) (GFAP) from

Chemicon for astrocytes (1 : 30), and anti-(human

leuko-cyte antigen-DR) (HLA-DR) from Dako for microglia

(1 : 500)

Slides were then rinsed in NaCl⁄ Pi with 1% Tween-20

three times for 5 min, and then incubated in preadsorbed

mouse secondary antibody for 1 h Cell types were detected

using the Vectastain ABC kit (Vector Laboratories,

Burlin-game, CA), using diaminobenzidine as a substrate,

accord-ing to the manufacturer’s protocols Immediately followaccord-ing

immunocytochemistry, slides were dehydrated in a 0.3 m

ammonium acetate series, and then dried and exposed to

film to estimate signal strength Slides were then dipped in

NTB2 autoradiography emulsion (Kodak, Rochester, NY),

and incubated at 4C until development In situ

hybridiza-tion was quantified on each specific cell type by counting

grain density on cells and subtracting background grain

density

Long and accurate (LA) RT-PCR

The synthesis of first-strand cDNA was performed using

the SuperScript preamplification system from Life

Technol-ogies One to three micrograms of total RNA per reaction

was reverse transcribed using oligo(dT) as the primer

About 2 lL of the 20 lL total volume of cDNA was used

per PCR reaction The LA RT-PCR method utilizes a

mix-ture of Taq polymerase and a small amount of a

proofread-ing polymerase, producproofread-ing a reaction mixture with greatly

increased product fidelity, yield, length and reproducibility

over either enzyme alone LA RT-PCR was performed

using a kit from Tamara Shuzo (TaKaRa Bio Inc.) and

conditions had been adjusted to ensure that results were in

a linear range and that a plateau had not been reached

Primers used were as follows: (a) human RCAN1 mRNA

isoform 1, consisting of exons 4, 5, 6, and 7) the forward

primer was 5¢-GACTGGAGCTTCATTGACTGCGAGA-3¢,

corresponding to bases 79–103 of exon 1 (bases 1–25 of the

short exon 1-containing isoform), and the reverse primer

was 5¢-ACCACGCTGGGAGTGGTGTCAGTCG-3¢,

cor-responding to bases 1–25 of exon 7; (b) human RCAN1

mRNA isoform 4, consisting of exons 1, 5, 6, and 7) the

forward primer was 5¢-AAGCAACCTACAGCCTCTTGG

AAAG-3¢, corresponding to bases 1–25 of exon 4, and the

reverse primer was the same primer used to amplify

iso-form 1; and (c) human GAPDH, for which the primers and

conditions were the same as previously described [8]

All DNA fragments produced by LA RT-PCR were veri-fied by sequencing, using an ABI Prism377 DNA sequencer (Perkin-Elmer, Waltham, MA) in our core facility

Acknowledgements The authors wish to acknowledge the generous support

of NIH⁄ NIA grant no AG 16256 Tissue for this study was obtained from the Alzheimer’s Disease Center Neuropathology Core, Keck School of Medicine, Uni-versity of Southern California, Los Angeles, CA, which

is funded by P59-AG05142, National Institute of Aging

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