Cloning and developmental expression of human 66 kd neurofilament protein

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular

Brain Research

29 (1995) 177-184

Short communication

Cloning and developmental

expression of human 66 kd neurofilament protein

Sai-On Chan, Fung-Chow Saul Korey Department

Chiu

*

of Neurology, F-140, Albert Einstein College of Medicine, Brom, NY 10461, USA Accepted

16 November

1994

Abstract The complete human 66 kd neurofilament cDNA was isolated and its sequence was determined. Both the DNA and the predicted amino acid sequences showed a high degree of homology to the rat NF-66. This was substantiated by cross hybridization between the human NF-66 probe and rat NF-66 mRNA. Single gene copy was suggested from Southern blot analysis. RNase protection assay indicated that NF-66 was expressed in human fetal brain as early as the 16th gestational week. On the other hand, NF-L message was not detected until the 20th gestational week. NF-M message was not detectable up to the 24th gestational week. Keywords:

Neurofilament;

cDNA sequence;

Single gene copy; Human fetal brain; NF-66; NF-L; NF-M

In addition to the neurofilament (NF) triplets, NF-L, -M and -H, and the 57 kd peripherin, a 66 kd protein (NF-66/a-internexin) represents a newly discovered form of mammalian neurofilament subunits [3,5,11]. Based on similarities in protein sequence and gene structure, NF-66 has been classified as a type IV intermediate filament, along with the NF triplets. All of the type IV neurofilaments are likely derived from a common ancestral gene through gene duplication [6,20,21]. During the development of hamster brain, low levels of NF-L and NF-M messages can be detected as early as embryonic day 12 [13]. These messages then increase dramatically within the first month of postnatal growth before declining to the adult level. The expression of NF-H, however, can only be detected postnatally. Interestingly, expression of two other intermediate filaments, peripherin and vimentin, shows a pattern opposite to the neurofilament genes. These findings suggest very specific roles of neurofilaments in the development of brain [13]. Unlike the NF triplets, which are found in both the CNS and PNS, NF-66 is expressed predominantly in the CNS in the adult [3,11] and this suggests a special

* Corresponding

author. Fax: (1) (718) 430-8790.

0169-328X/95/$09.50 0 1995 Elsevier SSDI 0169-328X(94)00268-1

Science

B.V. All rights

reserved

role of NF-66 in the normal functioning of central neurons. There is, however, relatively scarce information on the functions of this neurofilament, and its interactions with other neurofilaments during brain development. In the developing rodent, NF-66 protein was detectable in the mouse rhombencephalic tissue at E9.5 [7] and in the rat hind brain at El2 [ll]. In view of the developmental difference between rodent and human brain, we report here a study of expression of NF-66, -L and -M in human fetal brain during the second trimester. A cDNA encoding the full length human transcript was cloned and the protein sequence was deduced. NF-66 expression was also studied in two human neuroblastoma cell lines. The present study is part of an ongoing research program that has been approved by the Albert Einstein College of Medicine Committee on Clinical Investigations and the City of New York Health and Hospitals Corporation. Informed consent was obtained from all participants. Fetal tissue from abortuses of normal women was collected after elective pregnancy termination. The age of the abortuses was determined by multiple parameters including the date of the last menstrual period by history, uterine size by bimanual and abdominal examination, ultrasonography using predominantly the maximum biparietal diameter and, post-abortally, by measurement of fetal foot length. If a

178

S. -0. Ghan, F.-C. Chiu /Molecular

Brain Research 29 (1995) 177-184

1 10 20 30 40 50 TGTAGCTCGCGTTGAAGCCGCACGTCCGGCCCCGATCCCGGCACC~AGCTTCGGCTCG M S F

60 G

S

61 70 80 90 100 110 GAGCACTACCTGTGCTCCTCCTCCTCCTACCGCAAGGTGTGTTCGGGGATGGCTCTCGCCTG EHYLCSSSSYRKVFGDGSRL

120

121 130 140 150 160 170 TCCGCCCGCCTCTCTGGGGCCGGCGGCGCGGGGGCTTCCGTCGAGTCGCTGTCCCGCAGC S S A R L S GAGGAGASVESL

180

181 190 200 210 220 230 AATGTGGCCTCCTCGGCCGCCTGCTCCTCGGCCTCGTCGCTCGGCCTCGGACTGGCCTAT S L G L G L NV A S S AACSSAS

R

240 A

241 250 260 270 280 290 GCCCGGCCGCCGGCGTCCGACGGGCTGGACCTGAGCCAGGCGGCGGCGCGCACCAACGAG A A A R TN ARPPASDGLDLSQ 301 310 320 330 340 TACAAGATCATCCGCTCCAACGAGAAGGAGCAGCAGGCGACCGCTTCGCC SNEKEQ LQGLNDRFA Y K I I R

S

Y 300 E

350

360

361 370 380 390 400 410 GTGTTCATCGAGAAGGTGCATCAGCTGGAGACGCAGAACCGCGCGTTGGAGGCCGAGCTG NRALEAEL V F I E KVHQLETQ

420

421 430 440 450 460 470 GCGCGCTGCGACACCCACGCTGAGCCGTCGCGCGTCGGGCAGCTCTTCCAGCGCGAGCCT E P SRVGQL F Q R A R C D T H A

480 E

P

481 490 500 510 520 530 CGCCACCTGCCGCAGCTGGAGGAGGCCAGCTCGGCTCGCTCGCAGGCCCTGCTGGAGCGC SQALLER RHLPQLE EASSAR

540

541 550 560 570 580 590 GACGGGCTGGCGGAGGAGGTGCAGCGGCTGCGGGCGCGCTGCGAGGAGGAGAGCCGCGGA D G L A E E V Q R L RARCEEE S

600 R

G

601 610 620 630 640 650 CGCGAACGCCGAGCGCGCCTGAAGCGGCAGCAGCGCGACGTGGACGGCGCCACGCTGGCC RERRARL KRQQRDVDGATLA

660

661 670 680 690 700 710 CGCCTGGACCTGGAGFLAGAGGTGGAGTCGCTGCTGGACGAGCTGGCCTTCGTACGCCAG RLDLEKKVE SLLDELAFVRQ

720

?21 730 770 740 760 750 GTGCACGACGAGGAGGTAGCCGAGCTGCTGGCCACGCTGCAGCGGTCGTCGCA(~GCCG~G s Q V H D E E V A E L L A T L Q R S

780 h

A

781 790 800 810 820 830 GCCGAGGTGGACGTGACTGTGGCTAAACCAGACCTGACCGCGGCTCTGAGGGAGATCCGC A E VDVTVAKPDL TAALREIR

840

841 850 860 870 880 890 GCCCAGTATGAGTCCCTGGCCGCTAAGAACCTGCAGTCCGCGG~G~TGGTACAAGTCC AQYESLAA K N L Q S AEEWYKS

900

901 910 920 930 940 950 AAGTTTGCC~CCTGAACGAGCAGGCGGCGCGCACGACCGAGGCCATCCGGGCCAGCCGC KFANLNE QAARTTEAIRASR

960

961

970

980

990

1000

1010

102c

GACCAGATCCACGAGTATCGGCGCCTGCAGGCGCGCACCATCGAGATCGAGGGCCTGCGC DQIHEYRRLQARTIEIEGLR 1021 1030 1040 1050 1060 1070 GGGGCCAACGAGTCCTTGGAGAGGCAGATCCTGGAGCTGGAGGAGCGGCACAGTGCCGAG GANESLERQILELEERHSAE

108C

1081 1090 1100 1110 1120 1130 GTAGCTGGCTACCAGGATAGCATTGGGCAGCTGGAGAATGATCTGAGGAACACCAAGAGT VAGYQDSI GQLENDLRNTKS

1140

1141 1150 1160 1170 1180 1190 GAGATGGCACGCCACCTTCGGGAATACCAGGACTTGCTCAATGTCAAAATGGCTCTTGAC E M A R H L R E Y QDLLNVKMALD

1200

S.-O. Chan, F.-C. Chiu /Molecular

Brain Research 29 (1995) 177-184

1250 1230 1240 1210 1220 1201 ATTGAGATAGCAGCTTACAGGAAACTGCTGGMGGCGAGGAGACACGTTTTAGCACCAGT I E I A A Y R T R'F S KLLEGEE

179 1260

T

S

1310 1290 1300 1270 1280 1261 GGGTT~GCATTTCGGGGCTGAATCCACTTCCCAATCCAAGTTACCTGCTCCCACCTAGA G L S I SGLNPLPNPSYLLPPR

1320

1370 1350 1360 1330 1340 1321 ATCCTCAGTGCTACAACCTCCAAAGTCTCATCCACTGGGCTATCACTTAAGAAAGAGGAG SLKKEE SATTSKV S S TG L I L

1380

1430 1410 1420 1390 1400 1381 GAGGAGGAGGAGGCATCTAAGGTAGCCTCTAAGAAAACCTCCCAGATAGGGG~GTTTT SKVASKKTSQIGESF E E E E A

1440

1490 1470 1480 1450 1460 1441 GAAGAAATATTAGAGGAGACAGTAATATCTACTAAGAAAACCGAGAAATCAAATATAGAA S N EEILEETVISTKKTEK

1500 I

E

1550 1530 1540 TAmTTCCATTGCTTTGAAAAAGTTAATGCTTM

1560

1610

1620

1670 1650 1660 1630 1640 1621 CCACTATAAFlATGTCTTCAAGGCTTCAGTCTCATATTTAGTATTGAATACTTACATTCTC

1680

1730

1740

1790 1770 1780 1750 1760 1741 TATCTAAGAATACAGCTTTCTGACCTTCAGCTCACGTTCACAGTGATCGATGATTCAGGT

1800

1850 1830 1840 1810 1820 1801 GCAGAGGAAGTACAAACTAAGGTGCTAAATCTGAGATCATCGTCATTTGCTGTGAACTGA

1860

1910 1890 1900 1870 1880 1861 AATTAAAACTATTCATGCTACCCAGCCATTACCCAGCTAAATAATCTTACTCTAGATACC

1920

1950 1960 1930 1940 1970 1921 TAAAACATAAGATCACTGCCAGAGATAAACT~TGGTCCACACCCAATTCCACTCTGATA

1980

2030 2010 2020 1990 2000 1981 GAATTATTTCAC~TAATAGGTGTTTGTTTAATGGACACTTTTCACCTCCTTCAATTCC

2040

1510 1520 1501 GAAACCACCATTTCAAGCCAAMUU ETTISS Q K

1

1590 1570 1580 1561 GAGGGAATGATATGCATTTGACTTGTTAAACAGCCTATTC

1600

1710 1720 1690 1700 1681 TAATAAGAAAACCACCCCTTAGATTGAAGTAAACTGCAGTTACAGATGAGT

2050 2060 2041 ATATATCCTTTTCTTCTATGTAGG-

2070 2090 2080 TAGTCTAGTGTAGTATTCTTCCCTTTTAA

2010

2150 2130 2140 2110 2120 2011 ACACATTTTGGTTCTTGCTCAAAAGAACTTACCCCCATTCGCGCTCTGCTCAGTGGGTAA

2160

2190 2200 2170 2180 2210 2161 AATTAGATGCATGTTGACCATGTCTATGATTCGTGT~TTACTTATCCCTGCCCATTTCA

2220

2230 2240 2221 TATTCTTTCAATTCTGTAGGTTAAMAAA

2250 2260 2270 TGGCAGTGATGAATTTTAAGGGTTTCCCCCA

2310 2290 2300 2281 2330 2320 ACATATAAAATAATGCAAAGAATCCCTAGACTTTAGGCTTTCAGCTTACACAAATCTATT 2350 2341 TAATTAGGAAAAAAA

2370 2380 2360 2390 CTATTAAGGATGTAGCTTTTCACCTTTACTTTAGAAGCACAGTAA

2280

2340

2400

2430 2410 2420 2401 2450 2440 ATATCCCAAACTGTGATGAAGCCGACCTTAGATTTAGTAGTGTAAGCTAGAAGAAGTGAG

2460

2490 2470 2480 2461 2510 2500 TGTTTCTATGAGTGGAAAAAGCCAAGGTGTCATTTATGTGTCAGTTCATTTGTGGTGATA

2520

2550 2530 2540 2521 2570 2560 TAGAATTAGCTTTTTCATCCCCTTACCCTTAGAATCCACGTATTCAGCTTCCCACTAGCC

2580

2610 2590 2600 2581 2630 2620 TAGGTCATAAAGAGGCGTTGCTTTCACTCTCCTATGCCTTTTACGAGTGCT~GTGTAGG

2640

2670 2650 2660 2641 2680 ATATTTTGTCACCAGAACACAACTGTACCGCCCCAAAGTAG~CCACTTCTGGTCA

2700

2690

S.-O. Chan, F.-C. Chiu /Molecular

180

2701

2710

2720

Brain Research 29 (1995) 177-184

2730

2740

2750

2760

2810

2820

2870

2880

2930

2940

2990

3000

3050

3060

3110

3120

ATCfiACACTACCAGCGTATATATAAGAGACATCTTTCTCTTTTCTAAAAGACTTCCCT 2761

2770

2780

2790

2800

AACACTTACCCCATGGCTGCACAGTTGGTGGGGTCCTGCCGGAGAGGAAGAGACACTCAG 2821

2830

2840

2850

2860

ACCAGAGAAGGGGTGTGCATGCGCCTACTCGCTTGCTAGAGTAGATTCTGGACAGTCAG 2881

2890

2900

2910

2920

CTCTTCATCTGCCCAACTGTGTAGCATCTCGATTGCCAGTCTTCATGTGTGCC~GGCTG 2941

2950

2960

2970

2980

ATGCAGGATTTGTTCTCTGTCCAGCAGTCACTTCGGCCAGAGCTG~GAGTTGCC~CGTC

3001

3010

3020

3030

3040

TCTGTTCCATGTCTCCTTTAAGAGCTCTGGTGATAAGGACATGATGCTTTTACTGAACTT 3061

3070

3080

3090

3121

3130

3140

3100

AAAAATACTTTTCCTTACCTT

TCTTATCCTAGCACATGCTTCAATAGTTCAAGGAACTTG 3150

3160

3170

3180

TACCCCATCCCTGTCTTTACTCCTCACACTTACTGTAAGACATTAGTAACATAATAAATT 3181

3190

AAAAGTCACAGATCTCA

1

MSFGSEHYLCSSSSYRKVFGDGSRLSARLSGAGGAGA.SVESLSRSNVAS

1

/11/11111111/1/11/l I I l/Il/IIIIII MSFGSEHYLCSASSYRKVFGDGSRLSARLSGPGASGSFRSQSLSRS~AS

50

50 99

SAACSSASSLGLGLAYARPPASDGLDLSQiAARTNEYKIIRSNEKEQLQG

I

./Ill/lIIIIlIlll 51

49 I I I I I I I I I

Il/II/IlIIIIlIII/I//II

IIIlII/l 100

TAACSSASSLGLGLAYRRLPASDGLDLSQUARTNEYKIIRTNEKEQLQG

100

LNDRFAVFIEKVHQLETQNRALEAELAR.CDTHAEPSRVGQLFQREPRHL

101

LNDRFAVFIEKVHQLETQNRALEAELAALRQRHAEPSRVGELFQRELREL

149

.PQLEEASSARSQALLERDGLAEEVQRLRARCEEESRGRERRAR.LKRQQ

I I I I I I I I I I I I I I I I I I I I I I I I I I I

Ill/l///I

IIIIIIII

llllI/lI/lIlIII//IIIIIIIIllI

148 III/l

RAQLEEASSARAQALLERDGLAEEVQRLRARCEEESRGREGAERALKAQQ

197

RDVDGATLARLDLEKKVESLLDELAFVRQVHDEEVAELLATLQRSSQAAA

150 196

I

15i

I

I

II

II 200 246

/ll//lllllllllllllllllllll/llllllllllllllll

IllIll

201

RDVDGATLARLDLEKKVESLLDELAFVRQVHDEEVAELLATLQASSQAAA

250

247

EVDVTVAKPDLTAALREIRAQYESLAAKNLQSAEEWYKSKFANLNEQAAR

296

l/I/

II/Ii/l

Illl///lllllllIII/llIlIIIIIlll/lIIlIl

251

EVDVAVAKPDLTSALREIRAQYESLAAKNLQSAEEWYKSKFANLNEQAAR

300

297

TTEAIRASRDQIHEYRR.LQARTIEIEGLRGANESLERQILELEERHSAE

345

301

I I I I I I I I IllIll lIIl/llllllllllll/llllllI/III//l STEAIRASREEIHEYRRQLQARTIEIEGLRGANESLERQILELEERHSAE

350

346

VAGYQDSIG~LENDLRNTKiEMARHLREYQDLLNVKMALDIEIAAYRKLL

351

VAGYQDSIGQLESDLRNTKSEMARHLREYQDLLNVKMALDIEIAAYRKLL

396

EGEETRFSTSGLSISGLNPLPNPSYLLPPRILSATTSKVSSTGLSLKKEE

/lIIlIIIIl/l

395

Il//lllllllII/IllIlllllllll////lIIllI

IIlIllllIIIIl/ll1lllllIIl//I/lI//

IIIIIII

401

EGEETRFSTSGLSISGLNPLPNPSYLLPPRILSSTTSKVSSAGLSLKKEE

446

EEEE.....ASKVASKKTSQIGESFEEILEETVISTKKTEKSNIEE.TTI

451

I I I I I I I I I I I IIIIII Illll IlIIIlIl EEEEEEEEGASKEVTKKTSKVGESFEETLEETVVSTKKTEKSTIEEITTS

490

;SQKI*

494

501

I I I I SSQKM*

506

400 445 /IlIll/l 450 489 Ill

II 500

Fig. 1. Analysis of human NF-66 cDNA and amino acid sequence. Panel A shows the complete cDNA sequence for human NF-66. The predicted amino acid sequence is indicated. The initiation start site, translation termination site and polyadenylation signal are underlined. Panel B depicts an amino acid sequence comparison between human and rat NF-66 [S].

conflict arose between the various measures of gestational age, fetal foot length was accepted as the standard [9].

An oligo(dT)-primed hgtl0 fetal brain library (20-24 weeks old) and an oligo(dT) plus random-primed Agtll fetal brain library (20-24 weeks old) (Clontech, CO>

S.-O. Chan, F.-C. Chiu /Molecular

were used for screening the human NF-66 cDNA clones according to standard procedures [lo]. The probe used was a 2.8 kb cDNA clone that contained the full coding sequence of rat NF-66. The insert DNA from the phage clone was subcloned into pGEM-4Z (Promega) for dideoxy sequencing 1191 according to Sequenase protocol (United States Biochemicals) using [ 35S]dATP (DuPont). The sequenced DNA samples were resolved on a 6% polyacrylamide/7 M urea gel that was subsequently dried before exposure to Kodak XAR film. The MSN [18] and IM32K [12] neuroblastoma cells were cultured as described. Total RNA was isolated from human fetal forebrain and neuroblastoma cells by extraction with guanidium thiocyanate followed by centrifugation through a cesium chloride density gradient [ll. Northern blot analysis [l], Southern blot analysis [El and RNase protection assay [161 were performed as described. The pRNF66 plasmid that contained the full coding sequence for rat NF-66 was used to screen the human fetal library. pHNF661 and pHNF662, which together contained the full length cDNA for human NF-66, were used for Southern blot and Northern blot analyses. DNA was labelled by the random priming labelling kit from Boehringer Mannheim (Indianapolis, IN). We found that a polyclonal antibody generated against rat NF-66 protein can cross-react with human NF-66 protein (unpublished result). Thus, a high degree of sequence homology between the rat and human NF-66 genes was expected. A rat 2.8 kb NF-66 cDNA that contains the entire coding region was generated by a combination of PCR strategy and cDNA library screening (unpublished result) [5]. This rat NF-66 probe was used to select the NF-66 cDNA from two 20 to 24 week old human fetal brain libraries. Fig. 1A shows the complete sequence for the human NF-66 message. The ATG start site and the termination site are underlined. It should be noted that although the polyadenylation signal (AATAAA) is present, a poly A tail could not be detected. Since an oligo(dT)-primed AgtlO library was used for isolation of the 3’ untranslated region, it is likely that the poly A tail is very short or is lost during the library construction. The human NF-66 protein sequence showed homology of more than 90% with rat NF-66 (Fig. lB), indicating an evolutionary conservation. The deduced human NF-66 protein consisted of 494 amino acids and was 12 residues shorter than its rat counterpart. The major difference resided on the tail region of the protein. In contrast to rat NF-66, which contains 10 glutamic acid residues, only 6 glutamic acid residues were found in human NF-66. It has been shown that deletion of the tail region of rat NF-66 protein does not affect the ability of NF-66 to polymerize with other neurofilaments [2]. Similar findings were found for other neurofilaments such as NF-L and NF-M [8,22].

Brain Research 29 (1995) 177-184

181

23 kb9.4

-

6.6

-

A 2.3

-

2.0

-

1.35

-

26S-

B l6S--

Fig. 2. Southern and Northern blot analyses of human NF-66. Panel A represents the banding pattern of different restriction digests of 5 BamHI and Hind111 (lanes 1 to 3, pg human DNA by EcoRI, respectively). DNA was resolved on a 0.8% agarose gel. Panel B depicts the detection of NF-66 message in rat forebrain, human fetal brain of the 16th gestational week, MSN and IM32K cells (lanes 1 to 4, respectively). 10 +g of total RNA was resolved on a 1% agarose/2.2 M formaldehyde gel, and probed with the cDNA to human NF-66.

However, in other intermediate filaments such as desmin and vimentin, an intact tail region appears to be important for polymerization with other cytoskeletal proteins [4,17]. Both human and rat NF-66 messages contain a large 3’ untranslated region. The functional significance of this is unclear. There is some evidence, however, that an extended 3’ untranslated region may be associated with regulation of gene expression. For example, in the human interferon-beta transcript 1141, a c®ulatory sequence in the 3’ untranslated region governs the stability and translational efficiency of the message. The complete human NF-66 cDNA clone was used to probe several restriction digests of human DNA (Fig. 2A). Fragments of 6 kb and 4.4 kb were detected in the EcoRl digest, whereas fragments of 18 kb and 3.6 kb, and 2.2 kb and 1.4 kb were found for BamHl and Hind111 digests, respectively. The simple banding

S.-O. Chan, F.-C. Chiu/Molecular

182

pattern of restriction gested that the NF-66 the human genome.

digests for human NF-66 suggene was a single copy gene in

Brain Research 29 (19951 177-184

Expression of NF-66 in rat, human fetal brain and two tumor cell lines, MSN and IM32K, was also examined (Fig. 2B). Both rat and human samples showed a

A NFL

COII 2

Head

303 bp

NF66 Pst I

Pst I 1462 bp

429 bp

NFM Psi I

PSt

2746 bp

297 bD

B

M

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 M

1353bp

1078 872 603

281 271

-

Fig. 3. Developmental expression of human NF-66. Panel A is a diagrammatic representation of the design of each riboprobe used for the RNase protection assay. Panel B shows the expression of NF-66 (lanes 2 to 5), NF-L (lanes 7 to 10) and NF-M (lanes 12 to 15) in human fetal forebrain. s2P end-labelled @174 DNAs were used as markers (lanes M). Lanes 1, 6 and 11 represent the native ‘*P-labelled riboprobes for NF-66, NF-L and NF-M that have incorporated 58 bp, 58 bp and 51 bp of the pGEM-4Z (Promega) plasmid, respectively. For the RNase protection assay, 5 pg of total RNA of human fetal brain tissue from the 16th (lanes 2, 7 and 121, 18th (lanes 3, 8 and 13), 21st (lanes 4, 10 and 14) and 24th (lanes 5, 10 and 15) gestational week were used. In each assay, 100,000 cpm of ‘*P-labelled RNA probe were used.

S.-O. Chan, F.-C. Chiu /Molecular

message size of 3.2 kb, but with different intensities due to differences in DNA sequence between the two species (lanes 1 and 2, Fig. 2B). The rat message size is in close agreement with that reported [5]. NF-66 message can also be detected in the two tumor cell lines but at different intensities. Whereas NF-66 message is robustly expressed in MSN, it is barely detectable in IM32K. Equal loading was confirmed by stripping the blot and reprobing with cDNA to the 18s ribosomal gene (data not shown). Unlike the adrenergic MSN cells 1181, IM32K cells expressed both adrenergic and cholinergic phenotypes [12] and may represent a less differentiated stage. Since expression of neurofilaments is important for axon formation, NF-66 may serve as a useful marker for neuronal differentiation. To compare quantitatively in the same sample the levels of expression among NF-66, -L and -M, we used a RNase protection assay. We found that in human fetal brain, expression of NF-66 message (Fig. 3) and protein (unpublished data) were clearly detected as early as the 16th week of gestation, and increased rapidly and reached a steady level by the 18th week of gestation. In contrast, the expression of NF-L was barely detectable before the 18th gestational week and that of NF-M was not detectable throughout the gestational period of this study. In general, the expression of NF-66 is higher than that of NF-L. While it is difficult to compare human to rodent gestational ages, Kaplan et al. [ll] reported that expression of NF-66 could be detected as early as El2 in homogenates of rat brain, often preceding the expression of other neurofilament proteins. However, Kaplan et al. [ll] could not detect NF-66 protein by Western blotting or immunostaining in the forebrain region of El2 embryos. This differs from our findings since the autopsy tissue used in our study was derived from the frontal lobe of the forebrain. Furthermore, this discrepancy may be due to species differences or the low sensitivity of their antibodies. The functional roles of NF-66 in relation to other neurofilaments remain unresolved. Its pattern of expression during development and its distinct biochemical properties imply a specific role in neuronal differentiation. Transient expression of NF-66 in cells lacking any other intermediate filaments has shown that NF-66 protein can self-assemble, whereas none of the other neurofilaments can self-polymerize [2]. These data suggested at least two alternative roles for NF-66 in the morphogenesis of neurons. First, NF-66 may form an independent structural network without the involvement of other neurofilaments. This is consistent with the finding [3] that axonal fibers of cerebellar granule cells are immuno-positive for NF-66 only. Second, NF-66 may cooperate with NF-L to form the filamentous backbone to which NF-M and NF-H attach to form the cross-bridges. Transgenic mouse mod-

Brain Research 29 (1995) 177-184

183

els deprived of either NF-66 or NF-L potentially would address this function. It has been well-recognized that axons of the early developing nervous system are much smaller in diameter than their adult counterparts. The ability of NF-66 to self assemble and the absence of any carboxyl-terminal projection allow it to pack closely together, which is essential for maintaining small axonal diameter. This may partially explain the prevalence of this protein in early gestational periods. However, it remains possible that other intermediate filaments may copolymerize with NF-66 during early axonal formation. We thank Dr. William Lyman for his assistance in obtaining the brain tissue and Dr. Dennis Aquino for his comments and critical reading of the manuscript. This study was supported by USPHS NS23840 (F.C.C.), MH47667 (W.D. Lyman, Program Director) and NS23705 (W.T. Norton, Program Director). 111Chan,

S.O., Wong, S.S.C. and Yeung, D.C.Y., Regulation of Ki-ras gene expression in Reuber H35 cells, Eur. J. Biochem., 193 (1990) 681-686. 121Ching, G.Y. and Liem, R.K.H., Assembly of type IV neuronal intermediate filaments in neuronal cells in the absence of preexisting cytoplasmic intermediate filaments, J. Cell Biol., 122 (1993) 1323-1335. [31 Chiu, F.C., Barnes, E.A., Das, K., Haley, J., Socolow, P., Macaluso, F.P. and Fant, J., Characterization of a novel 66 kd subunit of mammalian neurofilaments, Neuron, 2 (1989) 14351445. H. and Franke, W.W., Assembly of a [41 Eckelt, A., Herrmann, tail-less mutant of the intermediate filament protein, vimentin, in vitro and in vivo, Eur. J. Cell Biol., 58 (1992) 319-330. El Fliegner, K.H., Ching, G.Y. and Liem, R.K.H., The predicted amino acid sequence of cy-internexin is that of a novel neuronal intermediate filament protein, EMBO J., 9 (1990) 749-755. [61 Fliegner, K.H. and Liem, R.K.H., Cellular and molecular biology of neuronal intermediate filaments, Intl. Rev. Cytol., 131 (1991) 109-167. V., Peng, D., Chiu, F.-C., and Van De 171 Galinovic-Schwartz, Water, T.R., Temporal pattern of innervation in the developing mouse inner ear: an immunocytochemical study of a 66-kD subunit of mammalian neurofilaments, J. Neurosci. Res., 30 (1991) 124-133. [81 Gill, S.R., Wong, P.C., Monteiro, M.J. and Cleveland, D.W., Assembly properties of dominant and recessive mutations in the small mouse neurofilament (NF-L) subunit, J. Cell Biol., 111 (1990) 2005-2019. 191 Hern, W.M., Correlation of fetal age and measurements between 10 and 26 weeks of gestation, J. Obstet. Gynecol., 63 (1984) 26-32. [lo] Huynh, T.V., Young, R.A. and Davies, R.W., Constructing and screening cDNA libraries in hgtl0 and hgtll. In D.M. Glover (Ed.), DNA Cloning: A Practical Approach, IRL Press, Oxford, Vol. I, pp. 49-78. [ll] Kaplan, M.P., Chin, S.S.M., Fliegner, K.H. and Liem, R.K.H., cY-Internexin, a novel intermediate filament protein, precedes the low molecular weight neurofilament protein (NF-L) in the developing rat brain, J. Neurosci., 10 (1990) 2735-2748. 1121 Ko, L.W., Sheu, K.F.R., Young, O., Thaler, H. and Blass, J.P., Expression in cultured human neuroblastoma cells of epitopes associated with affected neurons in Alzheimer’s disease, Am. J. Pathol., 136 (1990) 867-879.

184

S.-O. Chan, F.-C. Chiu /Molecular

[13] Kost, S.A., Chacko, K. and Oblinger, M.M., Developmental patterns of intermediate filament gene expression in the normal hamster brain, Brain Res., 592 (1992) 270-280. [14] Kruys, V.I., Wathelet, M.G. and Huez, G.A., Identification of a translation inhibitory element (TIE) in the 3’ untranslated region of the human interferon-beta mRNA, Gene, 72 (1988) 191-220. [15] Maniatis, T., Fritsch, E.F. and Sambrook, J., Molecular Cloning, Cold Spring Harbor, Laboratory, Cold Spring Harbor, NY, 1989, 9.31-9.58. [16] Maniatis, T., Fritsch, E.F. and Sambrook, J., Molecular Cloning, Cold Spring Harbor, Laboratory, Cold Spring Harbor, NY, 1989, 7.71-7.78. [17] Raats, J.M.H., Henderik, J.B.J., Verdijk, M., van Dort, F.L.G., Gerards, W.L.H., Bamaekers, F.C.S. and Bloemendal, H., Assembly of carboxy-terminally deleted desmin in vimentin-free cells, Eur. L Cell Biol., 56 (1991) 844103.

Brain Research 29 (1995) 177-184 [18] Reynolds, C.P., Biedler, J.L., Spengler, B.A., Reynolds, D.A., Ross, R.A., Frenkel, E.P. and Smith, R.G., Characterization of human neuroblastoma cell lines established before and after therapy, J. Natl. Cancer Inst., 76 (1986) 375-387. [19] Sanger, F., Coulson, A.R., Barrell, B.C., Smith, A.J.H. and Roe, B.A., Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing, I Mol. Biol., 143 (1980) 161-178. [20] Shaw, G., Neurofilament proteins. In R.D. Burgoyne (Ed.), The Neuronal Cytoskeleton, Alan R. Liss, New York, 1991, pp. 185-214. [21] Steinert, P.M. and Roop, D.R., Molecular and cellular biology of intermediate filaments, Annu. Rec.. Biochem., 57 (1988) 593625. [22] Wong, P.C. and Cleveland, D.W., Characterization of dominant and recessive assembly - defective mutations in mouse neurofilament NF-M, I Cell Biol., 111 (1990) 1987-2003.