Following fixation, tissue was dehydrated through ascending ethanol, embedded in paraffin, and 5 m sections were cut. == RNA Extraction and RT-PCR == RNA extraction and RT-PCR was performed as previously described TAPI-0 [25] with modifications. fluid prompted the hypothesis that hK3 may be produced in the brain. To test this notion, in this study we used RT-PCR amplification of brain tissue total RNA and examined hK3 protein by immunohistochemical, and immunoblot analysis. RT-PCR revealed several hK3 mRNA transcripts in the brain. Confirming these findings, both immunohistochemical staining and western immunoblotting showed evidence for hK3 protein in neuronal cells. Taken together, our findings support the expression of hK3 in neuronal cells reinforcing the concept of hK3 as a ubiquitous protein with more multifarious biological activity than previously believed. Ongoing research seeks to elucidate the functional significance of hK3 in brain TAPI-0 cells. Keywords:hK3, immunocytochemistry, mRNA, neuron, PCR, prostate specific antigen, protease, protein == Introduction == Human kallikrein-related peptidase 3 TAPI-0 (hK3), also known as prostate-specific antigen (PSA), is one of 15 serine proteases encoded within a 300-kb region of chromosome 19q13.3-4 [1], and its expression has shown to be primarily regulated by steroid hormones through androgen receptor-mediated transcription [2,3]. hK3, a chymotrypsin-like enzyme [4], is directly or indirectly linked to several biological functions including male fertility [5], cell proliferation regulation [6], and inhibition of angiogenesis [7]. The European Bioinformatics Institute currently recognizes 13 alternatively spliced mRNA transcripts of thehKLK3gene and isolation of additional variants is ongoing [812]. Characterization ofhKLK3splice variants and the alternative proteins they encode is incomplete [13], but their presence suggests a complex and diversein vivoactivity of hK3. Originally only three members of the serine protease family known as kallikreins were recognized. Of these,KLK1is transcribed in the telomere to centromere orientation, while the other two (KLK2,KLK3) are transcribed in the opposite orientation [14]. Now, there are 15 known kallikrein proteins encoded by tandemly located genes on chromosome 19 [15]. Although this family of genes shares a great deal of homology at the DNA and mRNA level [16], and have been shown to be comparably regulated by steroid hormones [17], they are differentially expressed throughout the body, including some in the brain. In this latter, high expression levels ofKLK5,KLK6andKLK8were measured in adult brain, andKLK8isoforms were differentially expressed in adult and fetal brains [18]. Also,KLK11was found in hippocampus and cerebellum with two alternatively spliced forms, one brain specific and one prostate specific. At the mRNA level, 12 of 15 kallikreins (excludingKLK3,KLK13andKLK15) have been found to be expressed in the CNS, and several have been linked to Alzheimer disease, Parkinsons disease and multiple sclerosis [19]. Once thought to be produced and secreted exclusively by prostatic epithelial cells [20], hK3 has since been shown to be neither organ, tissue, nor gender specific [21,22]. In fact, hK3 is detected in cerebrospinal fluid [23] where it was hypothesized that the protein may have originated from brain tissue. In partial support of this notion, hK3 expression was discovered and characterized in neuroblastoma cell lines SK-N-BE-2 and SKN-MC [24]. However, it is unclear if hK3 is producedin vivoin the brain, and if so, from what cell types. To address this issue, in this study we evaluated the presence of hK3 mRNA and protein in brain tissue by qualitative RT-PCR and immunohistochemical analyses. == Material and Methods == == Tissues == Using an approved IRB protocol, hippocampal or cortical tissue samples were obtained at autopsy (n = 8, ages 42-87, mean = 69) with post-mortem intervals (3.6-24 h, mean = 14) from the Alzheimer Disease Research Center at Case Western Reserve University and from the Cuyahoga County Coroner (SeeTable 1for details). As a positive control, surgical prostate tissue was also obtained (n = 3, ages 64-84, mean = 74). == Table 1. == Information on brain and prostate tissue used in this study For immunocytochemistry, brain samples were fixed in SOCS-2 either routine formalin or methacarn (methanol; chloroform; acetic acid; 6:3:1 v/v/v) at 4C overnight. Following fixation, tissue was dehydrated through ascending ethanol,.