Down-regulation of amyloid precursor protein by peptide nucleic acid oligomer in cultured rat primary neurons and astrocytes Linda Adlerz a , Ursel Soomets a,b , Linda Holmlund a , Sa ¨ de Viirlaid b , U ¨ lo Langel a , Kerstin Iverfeldt a, * a Department of Neurochemistry and Neurotoxicology, Stockholm University, SE-10691 Stockholm, Sweden b Department of Biochemistry, University of Tartu, 50411 Tartu, Estonia Received 12 September 2002; received in revised form 16 October 2002; accepted 18 October 2002 Abstract The amyloid precursor protein (APP) and its proteolytic cleavage products, the amyloid b peptides, have been impli- cated as a cause of Alzheimer’s disease. Peptide nucleic acids (PNA), the DNA mimics, have been shown to block the expression of specific proteins at both transcriptional and translational levels. Generally, the cellular uptake of PNA is low. However, recent studies have indicated that the effect of unmodified antisense PNA uptake is more pronounced in nervous tissue. In this study we have shown that biotinylated PNA directed to the initiator codon region of the APP mRNA (24– 111) was taken up into the cytoplasm of primary rat cerebellar granule cells and cortical astrocytes, using fluorescence and confocal microscopy studies. Uptake of PNA was faster in neurons than in astrocytes. Western blotting analysis showed that APP was strongly down-regulated in both neurons and astrocytes. Thus, unmodified PNA can be used for studies on the function of APP in neurons and astrocytes. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; Amyloid precursor protein; Antisense; Cerebellar granule cells; Peptide nucleic acid Processing of the amyloid precursor protein (APP) by b- and g-secretases results in the production of amyloid b peptides, the most prominent protein components of the plaques in Alzheimer’s disease brains. The physiological function is not clear, but APP has been suggested to be involved in neurite outgrowth, synaptogenesis, cell adhe- sion, synaptic plasticity, memory formation, and neuropro- tection against a broad number of insults (for review see Ref. [12]). Recently, it was suggested that APP may act as a receptor that signals through cleavage and nuclear trans- location of its cytoplasmic tail in an analogous manner to the Notch receptor [3,9]. Peptide nucleic acids (PNA) have received much atten- tion as possible antisense agents. PNA are DNA mimics with a polyamide backbone and were originally designed to bind to complementary dsDNA [11]. PNA has been shown to inhibit gene expression at translational and tran- scriptional levels. However, uptake of PNA by numerous cell types has been shown only at very high concentrations (20–100 mM) and after long incubation time (for recent review see Ref. [14]). Recently, it was shown that unmodi- fied PNA have pronounced antisense effects in neuronal tissue in vivo [15–18]. Earlier studies have indicated that PNA can enter neurons in culture, probably through an endocytotic mechanism, as suggested by a punctate distri- bution in the cytoplasm [1]. In this study we have investigated the uptake and anti- sense effect of biotinylated but otherwise unmodified, i.e. not coupled to a cell-penetrating peptide, PNA directed against APP mRNA in vitro in neuronal and astrocyte cell cultures using fluorescence microscopy, confocal micro- scopy and Western blotting. 15-mer PNA targeted to the initiator codon region of the rat APP mRNA (24– 111) with the sequence CTGGGCAG- CATCGTG was synthesized manually as described earlier [7]. The purity of PNA oligomer was 99% as analyzed by high pressure liquid chromatography. The molecular mass of the PNA was determined with a MALDI-TOF mass spec- trometer and the calculated value was obtained. Primary cultures of cerebellar granule cells (CGCs) were prepared as previously described [4]. The cells were plated at a density of 0.3 £ 10 6 cells/cm 2 in culture dishes or on Neuroscience Letters 336 (2003) 55–59 0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(02)01219-3 www.elsevier.com/locate/neulet * Corresponding author. Tel.: 146-8-164268; fax: 146-8- 161371. E-mail address: kerstin@neurochem.su.se (K. Iverfeldt).