Highly Specific and Sensitive Target Binding by the Humanized pS396-Tau Antibody hC10.2 Across a Wide Spectrum of Alzheimer's Disease and Primary Tauopathy Postmortem Brains.

BACKGROUND: Deposits of hyperphosphorylated tau fibrils are hallmarks of a broad spectrum of tauopathies, including Alzheimer's disease (AD). OBJECTIVE: To investigate heterogeneity of tau pathology across brain extracts from a broad selection of different tauopathies and examine the binding properties of the humanized pS396-tau antibody hC10.2 and six other anti-tau antibodies. METHODS: 76 individual tauopathy tissue samples were analyzed in a battery of assays: immunohistochemistry, ELISA, tau aggregation assay, western blot, [3H]PI-2620 and [3H]MK-6240 tau tracer binding, and aggregated seeding activity in RD_P301S HEK293T Biosensor cells. The efficiency of seven anti-tau antibodies to engage with pathological tau species was directly compared. RESULTS: Our data indicate that a strong correlation existed between the tau tracer binding, amount of tau aggregates, pS396-tau phosphorylation, and seeding activity. The hC10.2 antibody, which has entered clinical development, effectively engaged with its epitope across all individual cases of mid-stage and late AD, and primary tauopathies. hC10.2 was superior compared to other phospho- and total tau antibodies to prevent seeded tau aggregation in the biosensor cells. hC10.2 effectively depleted hyperphosphorylated and aggregated tau species across all tauopathy samples proportionally to the amount of tau aggregates. In AD samples, hC10.2 bound to ghost tangles which represent extracellular pathological tau species. CONCLUSION: S396 hyperphosphorylation is a feature of the formation of seeding-competent tau across different tauopathies and it is present both in intra- and extracellular pathological tau. hC10.2 represents an excellent candidate for a hyperphosphorylation-selective therapeutic tau antibody for the treatment of AD and primary tauopathies.

Anti-Aß Antibody Aducanumab Regulates the Proteome of Senile Plaques and Closely Surrounding Tissue in a Transgenic Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by accumulation of amyloid-ß (Aß) species and deposition of senile plaques (SPs). Clinical trials with the anti-Aß antibody aducanumab have been completed recently. OBJECTIVE: To characterize the proteomic profile of SPs and surrounding tissue in a mouse model of AD in 10-month-old tgAPPPS1-21 mice after chronic treatment with aducanumab for four months with weekly dosing (10 mg/kg). METHODS: After observing significant reduction of SP numbers in hippocampi of aducanumab-treated mice, we applied a localized proteomic analysis by combining laser microdissection and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the remaining SPs in hippocampi. We microdissected three subregions, containing SPs, SP penumbra level 1, and an additional penumbra level 2 to follow the proteomic profile as gradient. RESULTS: In the aducanumab-treated mice, we identified 17 significantly regulated proteins that were associated with 1) mitochondria and metabolism (ACAT2, ATP5J, ETFA, EXOG, HK1, NDUFA4, NDUFS7, PLCB1, PPP2R4), 2) cytoskeleton and axons (ADD1, CAPZB, DPYSL3, MAG), 3) stress response (HIST1H1C/HIST1H1D, HSPA12A), and 4) AßPP trafficking/processing (CD81, GDI2). These pathways and some of the identified proteins are implicated in AD pathogenesis. Proteins associated with mitochondria and metabolism were mainly upregulated while proteins associated with AßPP trafficking/processing and stress response pathways were mainly downregulated, suggesting that aducanumab could lead to a beneficial proteomic profile around SPs in tgAPPPS1-21 mice. CONCLUSION: We identified novel proteomic patterns of SPs and surrounding tissue indicating that chronic treatment with aducanumab could inhibit Aß toxicity and increase phagocytosis and cell viability.

Highly specific and selective anti-pS396-tau antibody C10.2 targets seeding-competent tau.

INTRODUCTION: The abnormal hyperphosphorylation of the microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD) and other tauopathies. METHODS: Highly specific and selective anti-pS396-tau antibodies have been generated using peptide immunization with screening against pathologic hyperphosphorylated tau from rTg4510 mouse and AD brains and selection in in vitro and in vivo tau seeding assays. RESULTS: The antibody C10.2 bound specifically to pS396-tau with an IC50 of 104 pM and detected preferentially hyperphosphorylated tau aggregates from AD brain with an IC50 of 1.2 nM. C10.2 significantly reduced tau seeding of P301L human tau in HEK293 cells, murine cortical neurons, and mice. AD brain extracts depleted with C10.2 were not able to seed tau in vitro and in vivo, demonstrating that C10.2 specifically recognized pathologic seeding-competent tau. DISCUSSION: Targeting pS396-tau with an antibody like C10.2 may provide therapeutic benefit in AD and other tauopathies.

Early depletion of CA1 neurons and late neurodegeneration in a mouse tauopathy model.

Alzheimer's disease (AD) and tauopathies, such as frontotemporal dementia (FTD), are characterized by formation of neurofibrillary tangles consisting of hyperphosphorylated tau. Further neuropathological characteristics include synaptic loss, neurodegeneration and brain atrophy. Here, we explored the association between hyperphosphorylated tau species, brain atrophy, synaptic and neuronal loss in a mouse model (rTg4510) carrying the human tau (hTau) P301L mutation found in a familiar form of FTD. We established that hTau expression during the first 6 postnatal weeks was important for the progression of tauopathy in rTg4510 mice. Short term suppression of postnatal hTau expression delayed the onset of tau pathology by approximately 6months in this model. Early postnatal hTau expression was detrimental to CA1 neurons of the hippocampus and reduced neuronal numbers in 6-10weeks young rTg4510 mice prior to the appearance of hyperphosphorylated hTau species in the hippocampus. Hyperphosphorylated hTau species emerged from 10 to 24weeks of age and were associated with increased ubiquitin levels, gliosis, and brain atrophy and preceded the synaptic loss and CA1 neurodegeneration that occurred at 48weeks of age. We present two consequences of hTau expression in CA1 in rTg4510 mice: an early decrease in neuron number already established prior to the presence of hyperphosphorylated tau species and a later neurodegeneration dependent on hyperphosphorylated tau. Neurodegeneration and synaptic protein loss were completely prevented when hTau expression was suppressed prior to the appearance of hyperphosphorylated tau species. Suppression of hTau expression after the onset of tau hyperphosphorylation and tangle pathology initiated at 16weeks partially rescued neuronal loss at 48weeks of age, while a reduction of neurodegeneration was no longer possible when hTau suppression was introduced as late as at 24weeks of age. Our results in rTg4510 mice argue that it is promising to lower hyperphosphorylated tau species at early stages of tau pathology to protect from neurodegeneration.

Dogs with Cognitive Dysfunction as a Spontaneous Model for Early Alzheimer's Disease: A Translational Study of Neuropathological and Inflammatory Markers.

Aged companion dogs with canine cognitive dysfunction (CCD) spontaneously develop varying degrees of progressive cognitive decline and particular neuropathological features correspondent to the changes associated with Alzheimer's disease (AD) in humans. The aim of the present study was to characterize certain aspects of neuropathology and inflammatory markers related to aging and CCD in dogs in comparison with human AD. Fifteen brains from aged dogs with normal cognitive function, mild cognitive impairment, or CCD were investigated and compared with two control brains from young dogs and brain sections from human AD subjects. The neuropathological investigations included evaluation of amyloid-ß (Aß) plaque deposition (N-terminally truncated and pyroglutamyl-modified Aß included), tau pathology, and inflammatory markers in prefrontal cortex. Cortical Aß deposition was found to be only of the diffuse subtype as no dense-core or neuritic plaques were found. The Aß deposition followed a progressive pattern in four maturation stages. Accumulation of the Aß peptide was also observed in the vessel walls. Both immunohistochemically and biochemically measured levels of Aß pathology in prefrontal cortex showed a consistent positive correlation to age but not to cognitive deficit severity. No evidence of neurofibrillary tau pathology was found. The level of pro-inflammatory cytokines was generally low and showed no significant association to cognitive status. The findings of the present study support the senescent dog with spontaneous cognitive dysfunction as a valuable non-transgenic model for further investigations of the molecular events involved in the neurodegenerative processes associated with aging and early stage AD, especially the Aß-related pathology.

Hyperactivity with Agitative-Like Behavior in a Mouse Tauopathy Model.

Tauopathies, such as Alzheimer's disease (AD) and frontotemporal dementia (FTD), are characterized by formation of neurofibrillary tangles consisting of hyperphosphorylated tau. In addition to memory loss, patients experience behavioral symptoms such as agitation, aggression, depression, and insomnia. We explored the behavioral phenotype of a mouse model (rTg4510) carrying the human tau P301L mutation found in a familial form of FTD. We tested these mice in locomotor activity assays as well as in the Morris water maze to access spatial memory. In addition to cognitive impairments, rTg4510 mice exhibited a hyperactivity phenotype which correlated with progression of tau pathology and was dependent on P301L tau transgene expression. The hyperactive phenotype was characterized by significantly increased locomotor activity in a novel and in a simulated home cage environment together with a disturbed day/night cycle. The P301L-tau-dependent hyperactivity and agitative-like phenotype suggests that these mice may form a correlate to some of the behavioral disturbances observed in advanced AD and FTD.

Altered function of hippocampal CA1 pyramidal neurons in the rTg4510 mouse model of tauopathy.

The formation of neurofibrillary tangles from the assembly of hyperphosphorylated tau leads to dendritic and axonal instability, synaptic degeneration, and neuronal loss. To understand the early physiological consequences of aberrant tau expression, we characterized the physiology of CA1 pyramidal neurons in rTg4510 female mice and non-transgenic (wt) littermate controls. We studied mice at the age of 10-12 weeks where only minimal hyperphosphorylated pretangle tau was present, and 22-24 weeks old mice with significant neurofibrillary tangle pathology. Our electrophysiological analysis included input-output relation, paired-pulse facilitation, and whole cell patch-clamp recordings of neurons to measure action potential threshold and action potential properties, chord-conductance, and characterization of AMPA receptor mediated synaptic transmission. We found that the input-output relation in field (excitatory postsynaptic potentials, EPSP) and whole cell recordings (excitatory postsynaptic currents, EPSC) were impaired in rTg4510 mice compared to wt controls at both ages. We measured a diminished tail current charge after depolarizing voltage input in rTg4510 mice compared to wt in both young and aged mice. Additionally, mini-EPSC properties (peak and decay time) were essentially similar between genotypes and age groups investigated. Surprisingly, in the 22-24 week old group, the mini-EPSC frequency was significantly increased (interevent interval 0.8 ± 0.1 in wt compared to 0.3 ± 0.1 in rTg4510 mice). These data indicate that the developmentally regulated expression of human P301L tau in CA1 pyramidal neurons coincide with changes in neuronal excitability but also that significant presynaptic changes occur late during the progression of tau pathology in this mouse model.

Pharmacological characterization of social isolation-induced hyperactivity.

RATIONALE: Social isolation (SI) of rats directly after weaning is a non-pharmacological, non-lesion animal model based on the neurodevelopmental hypothesis of schizophrenia. The model causes several neurobiological and behavioral alterations consistent with observations in schizophrenia. OBJECTIVES: In the present study, we evaluated if isolated rats display both a pre-pulse inhibition (PPI) deficit and hyperactivity. Furthermore, the sensitivity of SI hyperactivity to antipsychotic was evaluated. METHODS: Rats were socially isolated or group-housed for 12 weeks starting on postnatal day 25. In one batch of animals, the PPI and hyperactivity response were repeatedly compared. Furthermore, we investigated the robustness of the SI-induced hyperactivity by testing close to 50 batches of socially isolated or group-housed rats and tested the sensitivity of the assay to first- and second-generation antipsychotics, haloperidol, olanzapine, and risperidone, as well as the group II selective metabotrobic glutamate receptor agonist (LY404039). RESULTS: Socially isolated rats showed a minor PPI deficit and a robust increase in hyperactivity compared with controls. Furthermore, SI-induced hyperactivity was selectively reversed by all antipsychotics, as well as the potential new antipsychotic, LY404039. CONCLUSION: SI-induced hyperactivity was more pronounced and robust, as compared with SI-induced PPI deficits. Furthermore, SI-induced hyperactivity might be predictive for antipsychotic efficacy, as current treatment was effective in the model. Finally, using LY404039, a compound in development against schizophrenia, we have shown that the hyperactivity assay is sensitive to potential novel mechanisms of action. Thus, SI-induced hyperactivity might be a robust and novel in vivo screening assay of antipsychotic efficacy.

Socially isolated rats exhibit changes in dopamine homeostasis pertinent to schizophrenia.

Post-weaning social isolation of rats produces an array of behavioral and neurochemical changes indicative of altered dopamine function. It has therefore been suggested that post-weaning social isolation mimics some aspects of schizophrenia. Here we replicate and extent these findings to include an investigation of prefrontal cortical dopamine dynamics using in vivo microdialysis. Social isolation for 12 weeks after weaning caused increased locomotor activity in response to novelty and amphetamine challenge. In vivo microdialysis experiments revealed that while social isolation did not change basal dopamine levels in the nucleus accumbens, it did cause a significant reduction of basal dopamine release in the prefrontal cortex. In addition, social isolation lead to a significantly larger dopamine response to an amphetamine challenge, in both the nucleus accumbens and the prefrontal cortex compared to group housed controls. Taken together, these results indicate that post-weaning social isolation alters dopaminergic function with changes resembling subcortical hyperdopaminergia and prefrontal hypodopaminergia similar to what has been observed in schizophrenic patients.

Increased dopaminergic activity in socially isolated rats: an electrophysiological study.

The development of animal models mimicking symptoms associated with schizophrenia has been a critical step in understanding the neurobiological mechanisms underlying the disease. Long-term social isolation from weaning in rodents, a model based on the neurodevelopmental hypothesis of schizophrenia, has been suggested to mimic some of the deficits seen in schizophrenic patients. We confirm in the present study that socially isolated rats display an increase in both spontaneous and d-amphetamine-induced locomotor activity, as well as deficits in sensorimotor gating as assessed in a pre-pulse inhibition paradigm. In addition, in vivo electrophysiological studies revealed changes in dopaminergic cell firing activity in the ventral tegmental area of isolated rats when compared to group-housed controls. These alterations include an increase in the number of spontaneously active dopaminergic neurons, and a change of firing activity towards a more irregular and bursting firing pattern. Taken together, our findings suggest that the behavioral phenotype induced by social isolation may be driven by an overactive dopamine system.

Stereological brain volume changes in post-weaned socially isolated rats.

Rearing rats in isolation after weaning is an environmental manipulation that leads to behavioural and neurochemical alterations that resemble what is seen in schizophrenia. The model is neurodevelopmental in origin and has been used as an animal model of schizophrenia. However, only a few studies have evaluated the neuroanatomical changes in this animal model in comparison to changes seen in schizophrenia. In this study, we applied stereological volume estimates to evaluate the total brain, the ventricular system, and the pyramidal and granular cell layers of the hippocampus in male and female Lister Hooded rats isolated from postnatal day 25 for 15 weeks. We observed the expected gender differences in total brain volume with males having larger brains than females. Further, we found that isolated males had significantly smaller brains than group-housed controls and larger lateral ventricles than controls. However, this was not seen in female rats. Isolated males had a significant smaller hippocampus, dentate gyrus and CA2/3 where isolated females had a significant smaller CA1 compared to controls. Thus, our results indicate that long-term isolation of male rats leads to neuroanatomical changes corresponding to those seen in schizophrenia.

MicroRNAs show mutually exclusive expression patterns in the brain of adult male rats.

BACKGROUND: The brain is a major site of microRNA (miRNA) gene expression, but the spatial expression patterns of miRNAs within the brain have not yet been fully covered. METHODOLOGY/PRINCIPAL FINDINGS: We have characterized the regional expression profiles of miRNAs in five distinct regions of the adult rat brain: amygdala, cerebellum, hippocampus, hypothalamus and substantia nigra. Microarray profiling uncovered 48 miRNAs displaying more than three-fold enrichment between two or more brain regions. Notably, we found reciprocal expression profiles for a subset of the miRNAs predominantly found (> ten times) in either the cerebellum (miR-206 and miR-497) or the forebrain regions (miR-132, miR-212, miR-221 and miR-222). CONCLUSIONS/SIGNIFICANCE: The results indicate that some miRNAs could be important for area-specific functions in the brain. Our data, combined with previous studies in mice, provides additional guidance for future investigations of miRNA functions in the brain.

Functional and immunochemical characterisation of different antibodies against the erythropoietin receptor.

Since it was discovered that the hematopoietic hormone erythropoetin (EPO) exerts neuroprotective effects in the CNS, many studies on the EPO receptor (EPOR) function and localisation in the CNS have been performed. For this purpose, commercially available anti-EPOR antibodies have often been applied. As the literature data on these antibodies show inconsistencies, we here systematically compared six frequently used, commercially available EPOR antibodies for different applications. Five of the antibodies appeared to specifically recognize recombinant rat and human EPOR in HEK293 cells by Western blotting, but the same antibodies yielded different and inconsistent results when human UT-7 cells or rat brain tissue were applied. Immunocytochemical staining of EPOR-transfected HEK cells only produced consistent results with three of the six antibodies. All antibodies stained neurons in rat brain sections, but with large differences in the staining pattern and only the C-20 EPOR antibody was found to label astrocytes. Since EPOR antibodies have been applied in several studies as EPOR antagonists, we further tested the antibodies for their capacity to functionally block the EPO-EPOR interaction in a cellular signalling system with STAT-5 phosphorylation as readout. Here, only the MAB307 antibody showed a partial effect at concentrations of 5-50 microg/ml.

Neuropilin-1 and VEGF correlate with somatostatin expression and microvessel density in ovarian tumours.

Vascular endothelial growth factor (VEGF), is an angiogenic growth factor, expressed more highly in malignant than benign ovarian tumours. Neuropilin-1, which can act as a VEGF receptor has been shown to be associated with tumour angiogenesis in some cancer systems. Somatostatin (SST), a potentially anti-angiogenic factor, acts via somatostatin receptors that are expressed in ovarian cancer. We used immunohistochemistry to demonstrate expression of Neuropilin-1 in 63 malignant and 35 benign ovarian tumours and compared it to VEGF, Flt, Flk, SST expression and tumour microvessel density (MVD). Neuropilin-1 was expressed in 34/63 malignant and 22/35 benign lesions. VEGF, Flt, Flk and SST were expressed more highly in the epithelium of malignant and the vessels of benign lesions. VEGF expression correlated with SST expression in the epithelium (p<0.001) and the vessels (p<0.001), this co-expression was confirmed by dual immunostaining. The MVD for malignant lesions was higher than benign (p<0.001) and positively correlated to epithelial VEGF expression (p=0.001) and negatively correlated to vascular VEGF expression (p=0.025). These results show that Neuropilin-1 is expressed in ovarian tumours and also show that VEGF and SST are co-expressed in the same tissue compartments raising the intriguing possibility that SST may be important in angiogenesis in ovarian cancer.

Derivatives of erythropoietin that are tissue protective but not erythropoietic.

Erythropoietin (EPO) is both hematopoietic and tissue protective, putatively through interaction with different receptors. We generated receptor subtype-selective ligands allowing the separation of EPO's bioactivities at the cellular level and in animals. Carbamylated EPO (CEPO) or certain EPO mutants did not bind to the classical EPO receptor (EPOR) and did not show any hematopoietic activity in human cell signaling assays or upon chronic dosing in different animal species. Nevertheless, CEPO and various nonhematopoietic mutants were cytoprotective in vitro and conferred neuroprotection against stroke, spinal cord compression, diabetic neuropathy, and experimental autoimmune encephalomyelitis at a potency and efficacy comparable to EPO.

Regional pattern of binding and c-Fos induction by (R)- and (S)-citalopram in rat brain.

Citalopram is a racemic mixture of two stereoisomers, (R)- and (S)-citalopram. Both enantiomers were radiolabelled and used for in vitro receptor autoradiography in rat brain. High levels of specific [3H](S)-citalopram binding were observed in the amygdala complex, substantia nigra, superior colliculus and central grey. No specific binding of [3H](R)-citalopram was observed in any brain regions examined. S-citalopram induced a significant increase in c-Fos positive cells in central amygdala and in the bed nucleus of the stria terminalis (BST), compared to vehicle controls. A similar pattern of c-Fos activation was observed with (RS)-citalopram, sertraline and paroxetine, while (R)-citalopram did not increase c-Fos expression in these areas. The high affinity binding and apparent neuronal activation by (RS)-citalopram is thus confined to the (S)-enantiomer.

Distribution and pharmacology of alanine-serine-cysteine transporter 1 (asc-1) in rodent brain.

A polyclonal antibody against the Na+-independent alanine-serine-cysteine transporter 1 (asc-1) was raised and the specificity of the antibody verified by Western blots performed on membranes prepared from HEK293 cells transiently transfected with the cloned murine asc-1. The antibody was then used to localize the transporter in the brain of two rodent species by using immunohistochemistry at the light and electron microscopical level. asc-1-immunoreactivity (asc-1-ir) was widely distributed throughout the mouse and rat brain. Areas with high levels of asc-1-ir included hypothalamus, the medial septal area, globus pallidus, entopeduncular nucleus, cingulate and retrosplenial cortices. Moderate asc-1-ir was observed in several areas including layers III and V of the neocortex, thalamus, nucleus accumbens, caudate putamen, bed nucleus of stria terminalis, all amygdaloid nuclei, hippocampus (CA1-CA3 and hilus of the dentate gyrus), as well as several brainstem nuclei. asc-1-ir was observed as punctuate staining consistent with varicosities matching neuronal cell bodies and dendritic fields. At the ultrastructural level, asc-1-ir was mainly confined to presynaptic terminals. Immunostaining in either glial cell bodies or perivascular sites was not observed and white matter was completely devoid of asc-1-ir. Furthermore, the pharmacology of the Na+-independent uptake site for [3H]d-serine in rat brain synaptosomal P2 fractions was compared with the substrate specificity of the cloned human asc-1 transporter and a high degree of correlation was demonstrated. We conclude that asc-1-ir is widespread in the brain and limited to neuronal structures and that asc-1 may contribute to synaptic clearance of d-serine in brain.

Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo.

Erythropoietin (EPO) is a tissue-protective cytokine preventing vascular spasm, apoptosis, and inflammatory responses. Although best known for its role in hematopoietic lineages, EPO also affects other tissues, including those of the nervous system. Enthusiasm for recombinant human erythropoietin (rhEPO) as a potential neuroprotective therapeutic must be tempered, however, by the knowledge it also enlarges circulating red cell mass and increases platelet aggregability. Here we examined whether erythropoietic and tissue-protective activities of rhEPO might be dissociated by a variation of the molecule. We demonstrate that asialoerythropoietin (asialoEPO), generated by total enzymatic desialylation of rhEPO, possesses a very short plasma half-life and is fully neuroprotective. In marked contrast with rhEPO, this molecule at doses and frequencies at which rhEPO exhibited erythropoiesis, did not increase the hematocrit of mice or rats. AsialoEPO appeared promptly within the cerebrospinal fluid after i.v. administration; intravenously administered radioiodine-labeled asialoEPO bound to neurons within the hippocampus and cortex in a pattern corresponding to the distribution of the EPO receptor. Most importantly, asialoEPO exhibits a broad spectrum of neuroprotective activities, as demonstrated in models of cerebral ischemia, spinal cord compression, and sciatic nerve crush. These data suggest that nonerythropoietic variants of rhEPO can cross the blood-brain barrier and provide neuroprotection.

Targeting sst2A receptor-expressing cells in the rat hypothalamus through in vivo agonist stimulation: neuroanatomical evidence for a major role of this subtype in mediating somatostatin functions.

Numerous physiological studies as well as in situ hybridization and PCR experiments concur in reporting a role for the sst2A receptor in transducing somatostatin (SRIF) actions in the rat hypothalamus. However, the distribution of this receptor protein is not known within this structure. Regional and cellular localization of the sst2A receptor was therefore examined in the rat hypothalamus using highly sensitive immunohistochemical techniques. In close correspondence with the distribution of SRIF-immunoreactive fibers, numerous hypothalamic areas displayed sst2A receptor immunoreactivity. Receptor labeling was, however, diffusely distributed over the tissue, and few immunopositive cells were apparent. Unraveling the distribution of receptor-expressing cells was achieved through acute in vivo agonist stimulation and subsequent receptor internalization. At the cellular level, double-immunolabeling experiments with synaptophysin and microtubule-associated protein 2 demonstrated that sst2A receptors were predominantly internalized in perikarya and dendrites. Double-labeling experiments with SRIF revealed that 93% of arcuate, but only 18% of periventricular, SRIF-positive neurons expressed internalized receptors. Taken together, these results demonstrate for the first time that the sst2A receptor protein is widely, but selectively, distributed in the hypothalamus, and that postsynaptic sst2A auto- and heteroreceptors are well poised to play an important role in the somatostatinergic regulation of hypothalamic endocrine and metabolic processes.

Localization and mRNA expression of somatostatin receptor subtypes in human prostatic tissue and prostate cancer cell lines.

Somatostatin (SST) plays an important regulatory role in the physiological control of various organs including the prostate. Somatostatin receptors (SSTRs) and SST analogs are potential targets for prostate cancer treatment, especially since it has been shown that SST analogues are clinically effective in the treatment of advanced prostate cancer. The presence of SST containing neuroendocrine (NE) cells in the epithelium of the human prostate and their suggested role in the paracrine regulation of this gland prompted us to study the potential expression of somatostatin receptors (SSTRs) in human prostatic tissue and prostate cancer cell lines. Using the reverse transcriptase polymerase chain reaction (RT-PCR), we found the SSTR subtypes 1-3 in stromal cells and in prostate cancer cell lines LNCaP, PC-3 and DU 145. Immunohistochemical analysis of 27 radical prostatectomy specimens demonstrated the presence of hSSTR1 in a subpopulation of cancerous and NE cells, whereas hSSTR2 was found in the stroma, peritumoral blood vessels and tumor cells. Receptor subtype 3 was demonstrated to be present on the cell membrane of BPH and malignant areas. A strong immunoreaction (IR) of hSSTR4 was found in tumor cells, as compared with a less intense IR in adjacent BPH areas. Somatostatin receptor subtype 5 was not detectable. Western blot analysis revealed immunoreactive bands of molecular weight between 44-60 kDa. In summary, the present study clearly demonstrates the presence of hSSTR1-3 in tumoral and nontumoral epithelial cells as well as in the stromal compartment, whereas hSSTR4 was found to be confined to epithelial cells, and SSTR5 was not detectable.