Regional distribution and partial molecular characterization of CD4-related mRNA in human brain and peripheral tissues

Molecular Brain Research, 10 (1991) 23-31 Elsevier

23

BRESM 70276

Regional distribution and partial molecular characterization of CD4-related mRNA in human brain and peripheral tissues Jeffrey D. Erickson 1, John Q. Trojanowski 2 and Lee E. Eiden 1 1Unit on Molecular and Cellular Neurobiology, Laboratory of Cell Biology, NIMH/ADAMHA, Bethesda, MD (U.S.A.) and 2Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA (U.S.A.) (Accepted 6 November 1990)

Key words: CD4; 5"-Truncated CD4; mRNA expression; Macrophage/microglia; Human immunodeficiency virus type 1 (HIV-1); Acquired immune deficiency syndrome (AIDS); AIDS dementia complex

We purified human poly(A)÷ RNA from 11 individuals to assess the regional distribution of CD4 and CD4-related mRNA transcripts in human brain and in peripheral tissues by Northern blot hybridization. A 3.0 kb CD4 mRNA transcript was expressed in all brain areas and several peripheral tissues examined. A second CD4-related 1.8 kb mRNA species showed an uneven distribution in the brain with cortical regions possessing highest levels and basal ganglia, thalamus, cerebellum and spinal cord containing relatively lower amounts. Messenger RNA transcripts for CD8, a T cell specific marker, were not detectable in human brain by Northern analysis, yet were as abundant as CD4 in spleen. The expression of the 1.8 kb mRNA was tissue specific as it was not observed in peripheral tissues such as spleen, adrenal, colon, or lung, nor was it found in the choroid plexus, dorsal root ganglion and human neuronal (SY5Y) or astroglial (N132N1) cell lines. Blot hybridization and S1 nuclease protection analysis of poly(A)+ RNA with selective probes derived from CD4 indicated that the 1.8 kb mRNA transcript is truncated, lacking the extracellular protein coding region of CD4, and may in fact be a unique transcript from the CD4 gene locus rather than an alternatively spliced or processed CD4 mRNA.

INTRODUCTION C D 4 is the cell surface glycoprotein found on human m o n o c y t e - d e r i v e d cells and the helper/inducer lymphocyte subset of T cells mediating class II m a j o r histocompatibility c o m p l e x - d e p e n d e n t cellular i m m u n e recognition and function 8'36'38. It is also the r e c e p t o r utilized by the h u m a n immunodeficiency virus type 1 (HIV-1) to infect helper/inducer lymphocytes resulting in their selective reduction in the p e r i p h e r a l b l o o d of patients with acquired immunodeficiency s y n d r o m e ( A I D S ) 6'14"20'25. Cells of the bone m a r r o w - d e r i v e d m o n o c y t e lineage (macrophages/microglia) play a central role in HIV-1 pathogenesis as proviral sequences, R N A transcripts and/or p r o t e i n antigens are readily detected in these cells in brain, spinal cord, lung and o t h e r tissues of A I D S patients 9'13'21'35'37'4°'43. A direct C D 4 - d e p e n d e n t infection o f resident m o n o n u c l e a r inflammatory cells in these tissues by HIV-1 may play an i m p o r t a n t role in the d e v e l o p m e n t of the neurologic, p u l m o n a r y , and other complications of A I D S 5'19'3°'34'42. A n m R N A transcript of 3.0 kb, encoding the 432 amino acid CD4 protein, has been o b s e r v e d by N o r t h e r n

blot hybridization analysis in l y m p h o i d tissue and in h u m a n brain ~°'25. A second C D 4 - r e l a t e d m R N A transcript of a p p r o x i m a t e l y 1.9 kb has also b e e n d e t e c t e d with CD4 c D N A p r o b e s in h u m a n brain. These reports are inconsistent, however, regarding the distribution of h u m a n CD4 and C D 4 - r e l a t e d m R N A in the brain. In one study, the two C D 4 m R N A s were o b s e r v e d in p o l y ( A ) ÷ R N A from a single h u m a n c e r e b r a l cortex; the smaller C D 4 - r e l a t e d m R N A being far m o r e a b u n d a n t 25. O n the other hand, N o r t h e r n blot hybridization analysis of total R N A from a n o t h e r h u m a n c e r e b r a l cortex was negative for both CD4 m R N A s 1°. T h e 3.0 kb m R N A was found in subcortical areas such as the thalamus, pons and cerebellum while the shorter m R N A was o b s e r v e d only in one of two cerebellar samples from the same individual ~°. F o r these reasons, the s h o r t e r m R N A transcript in h u m a n brain has b e e n suggested to arise artifactually from cross-hybridization with residual 18S RNA18, 24. In the present study, we have purified p o l y ( A ) + R N A from eleven individuals to establish the presence, authenticity and regional distribution of C D 4 and CD4related m R N A in brain and p e r i p h e r a l tissues. N o r t h e r n

Correspondence: J.D. Erickson, Unit on Molecular and Cellular Neurobiology, Laboratory of Cell Biology, NIMH/ADAMHA, Building 36, Room 3A-17, Bethesda, MD 20892, U.S.A.

24 b l o t h y b r i d i z a t i o n a n d $1 n u c l e a s e p r o t e c t i o n a n a l y s i s i n d i c a t e d t h a t t h e 3.0 k b C D 4 m R N A is p r e s e n t in s e v e r a l p e r i p h e r a l tissues a n d in all b r a i n a r e a s e x a m i n e d w h i l e t h e 1.8 k b C D 4 - r e l a t e d m R N A

was b r a i n - s p e c i f i c a n d

e n r i c h e d in t h e c e r e b r a l c o r t e x .

MATERIALS AND METHODS

Tissues and cell lines Tissue samples were removed from 11 human cadavers 3.5-24 h postmortem, weighed, placed in aluminum foil, frozen in liquid nitrogen and stored at -70 °C. Adjacent samples were fixed in Bouin's solution, embedded in paraffin and examined microscopically to verify their location and to evaluate any abnormalities. The brain samples were mostly neuron-rich cerebral cortex and telencephalic nuclei but associated white matter was also present. The tissues were grossly and microscopically normal and the patients had no evidence of neurological disease. The H9 human lymphoma and SK-N-SH (SY-5Y) neuroblastoma cell lines were obtained from the American Type Culture Collection. The N132N1 human astrocytoma cell line was generously provided by Dr. Ken Harden (University of North Carolina). The H9 cells were grown in RPMI 1640 medium containing 10% fetal calf serum (Gibco). The neuronal and glial cell lines were maintained in Dulbecco's modified Eagles medium supplemented with 10% fetal calf serum. RNA isolation and Northern blotting Poly(A) ÷ RNA was purified by the guanidinium isothiocyanate/ cesium trifluoroacetic acid method followed by oligo-dT cellulose chromatography 2'29. 10-15 /~g of poly(A) ÷ RNA per lane were dissolved in 20/~1 of a solution containing 2.2 M formaldehyde/50% formamide/20 mM MOPS/5 mM sodium acetate/1 mM EDTA, pH 7.0/0.025% Bromophenoi blue and denatured at 60 °C for 10 min. The RNA was electrophoresed in 1.0% agarose gels containing 2.2 M formaldehyde/20 mM MOPS/5 mM sodium acetate/1 mM EDTA. The gels were stained in dilute ethidium bromide (4 gg/ml) for 15 min, rinsed in double-distilled water, destained for 16 h in electrophoresis buffer and photographed. After transfer to GeneScreen (New England Nuclear) nylon membrane and crosslinking by UV irradiation (120,000 ~J), the RNA blots were prehybridized (12 h) and then hybridized with human cDNA or oligonucleotide probes (106 cpm/ml) in a buffer containing 4x SSC (600 mM NaCl/60 mM sodium citrate, pH 7.0)/50% formamide/1 x Denhardts (0.5% ficoll, 0.5% polyvinylpyrrolidone, 0.5% bovine serum albumin)/200 ~tg/ml tRNA/250/ag/ml salmon sperm DNA (sheared and heat denatured) for 18 h at 42 °C. The filters were washed in 0.2 x SSC/0.1% sodium dodecyl sulfate (SDS) at 45-50 °C for oligonucleotides or 55 °C for cDNA probes and exposed to X-ray film with an intensifying screen at -70 °C. S1 nuclease protection Two overlapping 32p end-labeled CD4 probes were used to characterize the 1.8 kb CD4-related mRNA by S1 nuclease protection. Hybridization of 2-10 gg poly(A) ÷ RNA with 106 cpm probe (spec. act. approx. 108 cpm/~g) in 400 mM NaCI/1 mM Na2EDTA/40 mM PIPES (pH 6.4)/80% formamide was carried out in 50 #1 sterile glass microcapiilary tubes. The tubes were placed at 75 °C for 10 min to denature the double stranded DNA probes followed by hybridization at 52.5 °C for 8 h. Hybridization conditions were optimized for formation of D N A - R N A hybrids over D N A - D N A probe reannealing. The reactions were then snap frozen by transferring capillaries rapidly to a tray of dry ice. The contents of each capillary were expelled into 300/A of ice-cold S1 nuclease digestion mix containing 280 mM NaCI/4.5 mM zinc acetate/30 mM sodium acetate (pH 4.5)/10 /~g/ml salmon sperm DNA (sheared and heat denatured)/1000 U/ml $1 nuclease (Boeh-

ringer) and the digestion carried out at 45 °C for 30 min. The precipitated D N A - R N A hybrids were dissolved in alkaline loading buffer containing 50 mM NaOH/1 mM Na2EDTA/2.5% Ficoll/ 0.03% Bromophenol blue. Electrophoresis was performed in 1.5% agarose gels containing 50 mM NaCI/1 mM Na2EDTA with alkaline electrophoresis buffer consisting of 30 mM NaOH/1 mM Na2EDTA. Buffer was allowed to soak into the gel 30 min prior to loading. Following electrophoresis the gels were soaked in 7% trichloroacetic acid TCA for 30 min, dried under vacuum and exposed to X-ray film with an intensifying screen for 1 week at -70 °C. The double-stranded DNA probes (32p end-labeled only on antisense strand) were prepared from the 3000 bp CD4 cDNA inserted at the EcoRI site within the polylinker of the IBI 31 vector (International Biotechnologies Inc.). (a) The CD4-eontaining plasmid (pCD4) was restricted with BamHI which removed most of the 3" non-coding region retaining 290 bp of the 3" untranslated region and the entire 1452 bp of coding region. The linearized plasmid was dephosphorylated with calf intestinal phosphatase and then 5" end-labeled with D,-32p]dATP (5000 Ci/mmol; New England Nuclear) and T4 polynucleotide kinase (10 U, Biolabs Inc.). The 32p-labeled plasmid was then restricted with AhalII (cuts within the vector) and the 3zP-labeled 2717 bp BamHI-AhallI restriction fragment was separated and purified on glass beads (Geneclean) from a 1% agarose gel in 40 mM Tris-acetate/2 mM Na2EDTA. (b) The pCD4 was restricted at the AhalI site (CD4 cDNA base numbers 1352-1357) and the linearized plasmid was 3" end labeled with [a-32p]dCTP and the Klenow fragment of DNA polymerase (9 U, Promega). The a/P-labeled plasmid was restricted with BglI (cuts within the vector) and the 32p-labeled 3257 bp AhalI-BglI restriction fragment was separated and purified from a 1% agarose gel. The 3" probe contained 98 bp of coding region, the entire t548 bp of the 3" untranslated region and 1611 bp of the plasmid.

Oligo and cDNA probes Oligodeoxyribonucleotides (G + C content approximately 50%) complementary to the coding regions of human CD4 (cDNA base numbers: 33-mer, 1-33; 57-mer, 369-426; 34-mer, 739-772; 46-mer, 1376-1421), a 43-mer complementary to the coding region of human CD8 (cDNA base numbers: 693-735), and a 37-mer complementary to the coding region of human fl-actin (cDNA base numbers: 1466-1502) were synthesized on an Applied Biosystems 380A DNA synthesizer and purified by electrophoresis in a preparative denaturing acrylamide gel followed by low-melt agarose extraction. The oligonucleotide probes were labeled to a specific activity of 0.5-1 x 109 cpm/gg, using terminal deoxynucleotidyl transferase (100 U; Bethesda Research Labs) and [a-32p]dATP (3000 Ci/mmol; New England Nuclear). The full-length CD4 cDNA clone (3.0 kb) was generously provided by Dr. D.R. Littman (UCSF, CA) and was labeled to a specific activity of 0.5-1 x 108 cpm//~g with [a-32p]dCTP (3000 Ci/mmol; New England Nuclear) and E. coli DNA polymeruse I (5 U) using a nick translation kit (Amersham). The CD4 cDNA was also digested with BamHI and a 1 kb fragment (cDNA base numbers 1742-2742) purified by low-melt extraction and labeled by nick translation. RNA blots were rehybridized after stripping the previous probe with 95% formamide/1 mM EDTA/10 mM "Iris; pH 8.0 for 30 min at 60-65 °C followed by a thorough wash in 1 x SSPE (180 mM NaCl/2 mM EDTA/10 mM NaHzPO4; pH 7.4). Statistical analysis The relative abundance of CD4 and CD4-related mRNA in brain tissue was estimated by quantitating the bands appearing on the X-ray film by spectrophotometric scanning and normalizing the data from each sample to signals obtained with the fl-actin probe. The Wilcoxon rank sum test 33 was utilized to test the hypothesis that the ratio of 3.0 kb to 1.8 kb mRNAs in cortical areas (including hippocampus) was identical to that found in subcortical areas (including cerebellum). The relative abundance of fl-actin mRNA in total RNA (20/~g) from cortical and subcortical regions was not significantly different.

25 CASE # I HIp

Occ Thm

Cbl

Frn

Par

Tem

Cau

Spl

Put

3.0

CD4

1.8

1.9

13-actin

CASE # 2 Frn

Thm

Spi

Lng

Adr

Sgn

Cpx Cau

Put

Col 3.0

CD4

1.8

U[

Fig. 1. Northern hybridization analysis of mRNA from human brain regions and peripheral tissues. Approximately 2-15/zg of poly(A)+ RNA was prepared from each tissue sample: Hip, hippocampus; Occ, occipital pole; Thm, thalamus; Cbl, cerebellum; Frn, frontal lobe; Par, parietal cortex; Tern, temporal tip; Cau, caudate nucleus; SpI, spleen; Put, putamen; Spi, spinal cord; Lng, lung; Adr, adrenal gland; Sgn, sympathetic ganglion; Cpx, choroid plexus; Col, colon. The 3.0 kb CD4 cDNA and 46-mer oligonucleotide probes were hybridized to the RNA blots of case 1 (upper) and case 2 (lower), respectively, and both blots were stripped and reprobed with the fl-actin 37-mer probe. Exposure time was approximately 48 h for CD4 probes and 45 min for the fl-actin probe at -70 °C with an intensifying screen. The size of the CD4, CD4-related and fl-actin mRNAs (kb) are indicated on the right.

RESULTS

TABLE I

Northern blot analysis

areas

CD4 and CD4-related mRNA expression in cortical and subcortical

N o r t h e r n blot analysis of h u m a n p o l y ( A ) ÷ R N A indicated that the 3.0 kb CD4 m R N A transcript was expressed in all brain regions and in all p e r i p h e r a l tissues e x a m i n e d , albeit at low levels in the adrenal gland (Fig. 1). C D 4 m R N A expression in h u m a n brain was approximately 5% of that o b s e r v e d in spleen, calculated either by quantitating p o l y ( A ) ÷ R N A by ethidium staining, or with respect to the relative a b u n d a n c e of fl-actin in brain versus spleen R N A . The expression of the 1.8 kb C D 4 - r e l a t e d m R N A was brain specific as it was not o b s e r v e d in p e r i p h e r a l tissues such as spleen, adrenal, colon, or lung, nor was it found in the choroid plexus or dorsal r o o t ganglion (Fig. 1). Shorter exposures of X-ray films did not reveal the presence of the 1.8 kb m R N A in lung o r spleen samples that might have been obscured by the u p p e r band. H o w e v e r , the higher level of expression of C D 4 m R N A in lung and spleen (2/~g p o l y ( A ) ÷ R N A versus 15/zg from brain) m a d e it difficult to discern if very low levels of C D 4 - r e l a t e d m R N A might have been

Values represent the ratio of CD4 and CD4-related mRNA to fl-actin mRNA in cortical and subcortical areas from case 1 determined by spectrophotometric analysis of hybridization signals on X-ray films. The normalized data for each CD4 mRNA species are ranked from lowest to highest (in parenthesis) and summed within each cortical and subcortical group. Summed values ~<15 and 935 indicate significant differences with 2a = 0.02.

Cortical areas Frontal Temporal Occipital Parietal Hippocampus Suhcortical areas Thalamus Caudate Putamen Cerebellum

3.0 kb

1.8 kb

0.32 (6) 0.15 (2) 0.24 (4) 0.11 (1) 0.36 (7) (20)

0.71 (9) 0.70 (8) 0.56 (7) 0.50 (6) 0.49 (5) (35)

0.37 (8) 0.41 (9) 0.23 (3) 0.27 (5) (25)

0.19 (3) 0.17 (2) 0.20 (4) 0.16 (1) (10)

26 present. The CD4-related 1.8 kb m R N A transcript showed an uneven distribution in human brain with cortical regions (frontal lobe, temporal tip, parietal cortex, occipital pole, dentate gyms of the hippocampus) containing the highest levels and basal ganglia (head of caudate, p u t a m e n ) , thalamus (pulvinar), cerebellum (vermis) and spinal cord (thoracic level) contain relatively lower amounts. N o n - p a r a m e t r i c statistical analysis (Wilcoxon rank sum test) indicated that the distribution of the 1.8 kb CD4 m R N A between cortical and subcortical areas in the brain of case 1 was significantly different while the 3.0 kb CD4 m R N A was more evenly distributed (Table I). Qualitatively, a similar regional distribution of CD4 and C D 4 - r e l a t e d m R N A between cortex and telencephalic areas was observed in the brain of case 2 (Fig. 1). The 1.8 kb m R N A transcript was not observed

SP

8

÷

-

4

÷

÷

B.

in H9 or M O L T 4 human l y m p h o m a cell lines. Neither the 3.0 kb nor the 1.8 kb message were found in human peripheral neuronal (SY5Y) or astroglial (N132N1) cell lines (data not shown). There was significant variability between individuals in the relative expression of the 3.0 kb CD4 transcript and the 1.8 kb C D 4 - r e l a t e d m R N A in the cerebral cortex (Fig. 2). The 1.8 kb m R N A species was m o r e abundant than 3.0 kb CD4 m R N A in six of eleven cerebral cortex samples yet barely detectable in two cases (6 and 11). W h e n N o r t h e r n blots of h u m a n p o l y ( A ) + R N A were stripped and r e p r o b e d with a h u m a n CD8 oligonucleotide hybridization signals were o b s e r v e d only in the spleen with the CD4 to CD8 m R N A ratio equal to about 1. Low amounts (less than 10% of CD4 m R N A ) of CD8 m R N A were detected in lung (data not shown).

5

6

4-

7

4.

4.

C. CX)4

CI~

188

188

8

9

10

11

SIP

8

9

10

11

SP

Fig. 2. Hybridization specificity and variability of CD4 and CD4-related mRNA expression in human cerebral cortex. A: hybridization specificity of CD4 cDNA probe to poly(A)+ (2-15/~g) and poly(A)- (25/~g) RNA from spleen (SP) and cerebral cortex of cases 3-7. The temperature of the wash (0.2 x SSC/0.1% SDS, 55 °C) was reduced to 45 °C for cases 5-7 where slight non-specific binding to 18S ribosomal RNA can be observed in lanes of poly(A)- RNA. Additional samples of poly(A) ÷ RNA from spleen (2/~g) and cerebral cortex (10-15 ~g) from cases 8-11 were hybridized with CD4 (B) followed by CD8 (C) oligonucleotide probes, each washed under stringent conditions and exposed to X-ray film for 3 days.

27

A. Ahalll EcoR!

L-q ~L~

Aha[! BamHl

Extracellular _ ~

I

TM" (a) (b) (c)

Cytoplasmic

• • •

(d)

•

(e)

(f)

32p

(g)

32p

n.

C. 28s

2ss 3.0Kb

3.0Kb

18S

1.8Kb

18S

1.8Kb

Fig. 3. A: schematic representation of oligonucleotide and cDNA probes derived from 3.0 kb CD4 EcoRI insert: (a) 33-mer, (b) 57-mer, (c) 34-mer, (d) 46-mer, (e) 1 kb BamHI cDNA restriction fragment, (f) 2.72 kb S1 nuclease BamHI-AhalI restriction probe (includes 975 bp of plasmid), and (g) 3.26 kb S1 nuclease AhalI-BgllI restriction probe (includes 1611 bp of plasmid). Northern hybridization using first (B) a 1 kb BamHI fragment derived from much of the 3" non-coding region of the CD4 cDNA and then (C) a 57-mer oligonucleotide complementary to a 5" coding region of CD4 to human poly(A)÷ RNA from spleen (2/zg, left lane) and cerebral cortex (15/zg, right lane) of case 3.

Initially, oligonucleotides and cDNA fragments derived from CD4 were utilized to characterize the 1.8 kb CD4-related mRNA. As shown in Figs. 1 and 2 an oligonucleotide (46-mer; cDNA base numbers 13761421) complementary to the region coding for the cytoplasmic domain of CD4 recognized both CD4 mRNA species. In addition, the two CD4 mRNA species have homologous 3" non-coding regions as the 1 kb BamHI fragment (cDNA base numbers 1742-2742) also recognized both CD4 mRNA species (Fig. 3). However, an oligonucleotide (57-mer; cDNA base numbers 369 to 426) complementary to a 5" region of CD4 (V1 region) did not recognize the 1.8 kb CD4-related mRNA species in human brain (Fig. 3). Additional oligonucleotides derived from the 5" region of CD4 (33-mer cDNA base numbers 1-33; 34-mer cDNA base numbers 739-772) also did not recognize the 1.8 kb mRNA in human brain (data not shown).

S1 nuclease protection analysis In an effort to further characterize the human brainspecific CD4-related mRNA, S1 nuclease analysis using two overlapping CD4 cDNA probes to assess homology

between the 1.8 kb mRNA and the 3.0 kb mRNA, was conducted. A fragment of approximately 1742 bp was protected in poly(A) + RNA from H9 lymphoma cells, spleen and brain using a 2717 bp probe, which was 32p end-labeled at nucleotide 1742 (BamHI site) and complementary to the entire coding region of CD4 (1452 bp) including 290 bp of 3" non-coding region and 975 bp of plasmid (Fig. 4). Hybridization of this probe to brain poly(A) ÷ RNA protected an additional fragment of approximately 400 bp which was not observed in lymphoid RNA. No protection of the CD4 probe was seen with poly(A) ÷ RNA from human astroglial (N132N1) or neuronal (SY5Y) cell lines or with poly(A)- RNA (20 /zg). To determine whether the 400 bp protected fragment observed by S1 nuclease analysis of brain mRNA extended fully through the 3" end of the CD4 mRNA, we used a 3257 bp probe which was 32p end-labeled at nucleotide 1354 and complementary to the last 98 bp of coding region and including the entire 3" non-coding region of CD4 (1548 bp) and 1611 bp of plasmid. Hybridization of this probe protected a single fragment of approximately 1646 bp in poly(A) ÷ RNA from H9 cells, spleen and brain (data not shown). The S1 nuclease map

28

H9

Spl # 3

#4

A

N pA-

3.50

I .98 I .59

0.94

0.56

.4

0.14

Fig. 4. $1 nuclease mapping of CD4-related mRNA from human cerebral cortex. Solution hybridization of poly(A) ÷ RNA from H9 lymphoma cell line (1, 2, and 4#g), spleen (2 #g), brain (10-15/~g), astrocytoma (N132N1) cell line (20gg), neuronal (SYSY) cell line (20/~g) and poly(A)RNA (25 gg) from H9 cells with 32p end-labeled CD4 probe followed by $1 nuclease digestion to determine co-linearity of CD4-related mRNA to the 3.0 kb CD4 transcript. The relative size of protected cDNA (kb) was determined using markers of known MW prepared by HindIII/EcoRI double digests of lambda DNA.

of the 5" end of the CD4 m R N A species was performed two times with identical results, while the map of the 3" end was performed once. CD4 degradation artifacts of $1 nuclease analysis are observed in lymphoma cell RNAprotected c D N A when compared to cDNA protected by R N A from neuronal and astroglial cell lines, albeit at very low abundance (Fig. 4). In contrast to the brainspecific protection of approximately 400 bp of 5" cDNA from the 1.8 kb CD4-related m R N A no additional lower molecular weight CD4-related m R N A species are observed with lymphoid poly(A) + RNA by Northern blotting. DISCUSSION We have isolated poly(A) + RNA from brain and peripheral tissues of eleven cadavers to re-evaluate the

regional distribution of CD4 and CD4-related m R N A . Two m R N A species were detected in human brain poly(A) + R N A by blot hybridization with CD4 cDNA and specific oligonucleotide probes (Figs. 1 and 2). One of these transcripts was 3.0 kb and was identical to CD4 message found in the spleen; the second m R N A transcript was shorter and appeared to comigrate with 18S rRNA by Northern analysis. The smaller CD4-related m R N A does not represent cross-hybridization with residual 18S RNA: (1) hybridization with c D N A probes was observed only in lanes of poly(A) + RNA, and not in poly(A)- RNA (25 #g) under stringent (0.2x SSC/0.1% SDS; 55 °C) washing conditions, (2) the smaller CD4related m R N A (1.8 kb) could be separated from 18S rRNA (1.9 kb) by running agarose gels for longer periods of time, (3) an oligonucleotide (46-mer, G + C content = 50%) within the cytoplasmic coding region recognized

29 TABLE II Variable expression o f 3.0 kb relative to 1.8 kb CD4 mRNA in cerebral cortex

Ratios of the hybridization signals for the 3.0 kb and 1.8 kb mRNA species from each case were obtained from spectrophotometric analysis of the X-ray films. PMI, postmortem interval. Case

3.0/1.8 Age/sex PMI (h)

1

2

3

4

5

6

7

8

9

10

11

0.45 75/M 18

1.11 64/M 15

0.65 63/M 15

1.79 38/F 3.5

0.68 57/M 14

1.62 27/F 18

0.79 93/F 20

0.86 60/F 16

1.43 48/F 15

0.73 30/M 21

2.10 1/F 12

both CD4 m R N A species under stringent (0.2× SSC/ 0.1% SDS; 50 °C) washing conditions, and (4) solution hybridization of brain poly(A) + R N A with 32p endlabeled CD4 c D N A probes followed by $1 nuclease digestion protected two fragments corresponding to the 3.0 kb and the 1.8 kb m R N A (Fig. 4). HIV-1 proviral sequences, R N A transcripts and/or protein antigens have been detected in lymphoid tissue and cell lines, brain, skin, intestine and other tissues from AIDS patients 13'21'28'32'35'37'4°A3. Within the CNS, the primary HIV-l-associated pathology is also widespread and observed in all major subdivisions of the cerebral cortex, subcortical structures such as the basal ganglia, thalamus, rostral brain stem nuclei, spinal cord and in white matter 7'9A1'12'27'41. We found that the 3.0 kb CD4 m R N A was distributed in all brain regions and present in spinal cord, sympathetic ganglia, choroid plexus, colon, lung, and spleen samples (Fig. 1, Table I). The presence of CD4 m R N A in human tissues would support the hypothesis that AIDS encephalopathy and other complications are due to a primary infection by the AIDS retrovirus. It is unlikely that residual blood lymphocytes were responsible for the CD4 m R N A hybridization in non-lymphoid tissues. Blot hybridization of brain poly(A) + R N A to CD8, a T cell-specific marker, was not observed, yet the CD4 to CD8 m R N A ratio in spleen was about one (Fig. 2C). Even in lung, where blood perfusion is relatively high, the CD4 to CD8 m R N A ratio was much greater than one. It is likely that the CD4 m R N A detected in human tissues in the present study was derived from cells of the monocyte lineage. Macrophages are unique hematopoietic cells in that they develop a resident population in several tissues of the body 15'3t. CD4-dependent HIV-1 infection has been demonstrated in human peripheral macrophages and recently in primary brain microglial cultures 5"19. In addition, HIV-1 particles in brain, spinal cord and lung have consistently been detected in macrophage-derived cells. CD4-independent infection of peripheral neuroblastoma, glioblastoma and brain-derived glial cells, rhabdomyosarcoma, hepatocellular carcinoma and fibroblastoid cell

lines by HIV-1 has been reported 1"3'17'22"23. These infections are generally but not invariably non-productive and latent and require higher viral titres than lymphocyte and macrophage/microglial infection by HIV-1. The significance of in vitro infection of transformed cell lines in a CD4-independent manner is unknown as the corresponding parenchyma are apparently not infected or rarely infected in vivo in patients wih AIDS 39'43. We did not detect CD4 or CD4-related m R N A in neuronal or astrocytoma cell lines by either Northern blot analysis or $1 nuclease protection experiments. The expression of the 1.8 kb CD4-related m R N A appeared to be tissue specific; it was detected in brain but not in lymphoid or peripheral tissues. Within the brain the 1.8 kb m R N A species was more abundant in the cerebral cortical areas than in subcortical structures such as the basal ganglia, thalamus, cerebellum and spinal cord (Fig. 1, Table I). There was significant variability among several human cases in the relative expression of the 3.0 kb CD4 m R N A and the 1.8 kb CD4-related transcript in the cerebral cortex (Table II). The 1.8 kb m R N A species was more abundant than CD4 m R N A in six of eleven cerebral cortex samples yet barely detectable in cases 6 and 11 (Fig. 2). This m a y be due to differences in the amount of grey to white matter in tissue samples or to differences in m R N A stability during poly(A) + purification. However, similar ratios of 3.0/1.8 CD4 m R N A expression (<1) were observed in all cerebral cortical regions from case 1 (Table I). In case 2, where the cerebral cortex 3.0/1.8 m R N A ratio is greater than one, a similar enrichment of 1.8 kb m R N A is seen in cortical versus subcortical areas (Fig. 1). In addition, the 1742/400 bp cDNA ratio observed by $1 nuclease protection from the occipital pole of the cerebral cortex from cases 3 and 4 (Fig. 4) was similar to the 3.0/1.8 m R N A ratio observed by blot hybridization with poly(A) + R N A from the frontal lobe of these individuals (Fig. 2A). No obvious correlation between the 3.0 kb to 1.8 kb CD4 m R N A ratio and sex or postmortem interval (PMI) was observed (Table II). We are currently examining the possibility that the expression of the 1.8 kb

30 C D 4 - r e l a t e d m R N A may be developmentally specific and related to macrophage differentiation in the brain microenvironment. The relevance of the unique expression of the CD4-related m R N A is unclear at the present time. A CD4-related m R N A of approximately 2.3 kb has been observed in adult mouse brain t6"24'26. The fulllength 3.2 kb CD4 m R N A is not detected in the mouse CNS perhaps because murine monocyte/macrophagederived cells do not bear CD4 on their cell surface 4. The transcriptional start site of the 2.3 kb CD4-related m R N A characterized in mouse brain has been m a p p e d to nucleotide 845 of the CD4 c D N A by RNase protection and S1 nuclease analysis. A n A T G codon at nucleotide 890 of the mouse c D N A was p r o p o s e d to initiate the synthesis of a 154 amino acid cell surface region, together with the complete CD4 t r a n s m e m b r a n e and cytoplasmic regions. S1 nuclease protection experiments with p o l y ( A ) + R N A from human brain indicated that the 1.8 kb C D 4 - r e l a t e d m R N A is colinear with the distal 60% of the 3.0 kb CD4 m R N A , sharing the 3" non-coding region and possibly the cytoplasmic region and t r a n s m e m b r a n e domain, and none of the extracellular coding portion of CD4 (Fig. 4). The human 1.8 kb CD4-related m R N A would potentially encode a very different protein than that p r o p o s e d to exist in mouse brain. H o w e v e r , both human and mouse truncated m R N A species would encode proteins which lack the H I V binding epitope present in the extracellular region of CD4. The physiological or pathophysiological role (if any) of proteins expressed from CD4-related m R N A species in human or mouse brain is not known. It is doubtful that the human brain CD4-related m R N A arises by alternative splicing as a 33-mer oligonucleotide c o m p l e m e n t a r y to the 5" terminus of human CD4 m R N A did not recognize the 1.8 kb m R N A . The h u m a n brain 1.8 kb CD4-related m R N A is more likely to

arise via use of an alternate p r o m o t e r within the CD4 gene locus. In the mouse, a T A T A A e l e m e n t (within a CD4 exon) is positioned a p p r o x i m a t e l y 20 nucleotides upstream of the 2.3 kb brain m R N A transcriptional start site 16"24. While an equivalent T A T A A box is present at nucleotides 709-713 in the human CD4 c D N A , the potential initiating methionine found in mouse CD4 (amino acid residue 215) is not conserved 26. In addition, a human oligonucleotide ( c D N A base numbers 739 to 772) corresponding to the first 34 nucleotides of the mouse CD4-related m R N A transcriptional start site did not recognize the 1.8 kb m R N A in human brain. Further, S1 nuclease protection of p o l y ( A ) ÷ R N A from human brain did not include the region of CD4 c D N A which encodes any of the extraceUular domain. Thus, the human brain 1.8 kb C D 4 - r e l a t e d m R N A is clearly structurally different from the 2.3 kb mouse brain C D 4 - r e l a t e d m R N A . Like the mouse, however, the human brain C D 4 - r e l a t e d m R N A may arise via an alternative p r o m o t e r within the CD4 gene locus. This study definitively establishes the presence of CD4 and 5"-truncated CD4 m R N A s in h u m a n brain. The distribution of CD4 m R N A provides information on potential HIV-1 infection sites in h u m a n tissues. The relative expression of CD4 m R N A in the brain may be relevant to the variability of clinical s y m p t o m o l o g y observed in patients with A I D S dementia. The importance of the 5"-truncated CD4 m R N A in the h u m a n CNS awaits complete structural characterization of this m R N A and the protein in brain it potentially encodes.

Acknowledgements. The authors thank Dr. Michael J. Brownstein for synthesis of the oligodeoxyribonucleotidesand for critical review of the manuscript. We also thank the National Disease Research Interchange for additional human cerebral cortex tissue. The work by J.D.E. was in partial fulfillment of the Ph.D. degree requirement at The George Washington University, Washington DC and was supported, in part, by Genelabs Inc., Redwood City, CA.

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