Neurotensin Receptor Allosterism Revealed in Complex with a Biased Allosteric Modulator.

The NTSR1 neurotensin receptor (NTSR1) is a G protein-coupled receptor (GPCR) found in the brain and peripheral tissues with neurotensin (NTS) being its endogenous peptide ligand. In the brain, NTS modulates dopamine neuronal activity, induces opioid-independent analgesia, and regulates food intake. Recent studies indicate that biasing NTSR1 toward ß-arrestin signaling can attenuate the actions of psychostimulants and other drugs of abuse. Here, we provide the cryoEM structures of NTSR1 ternary complexes with heterotrimeric Gq and GoA with and without the brain-penetrant small-molecule SBI-553. In functional studies, we discovered that SBI-553 displays complex allosteric actions exemplified by negative allosteric modulation for G proteins that are Gα subunit selective and positive allosteric modulation and agonism for ß-arrestin translocation at NTSR1. Detailed structural analysis of the allosteric binding site illuminated the structural determinants for biased allosteric modulation of SBI-553 on NTSR1.

Catalytic site mutations confer multiple states of G protein activation.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) that function as molecular switches for cellular growth and metabolism are activated by GTP and inactivated by GTP hydrolysis. In uveal melanoma, a conserved glutamine residue critical for GTP hydrolysis in the G protein α subunit is often mutated in Gαq or Gα11 to either leucine or proline. In contrast, other glutamine mutations or mutations in other Gα subtypes are rare. To uncover the mechanism of the genetic selection and the functional role of this glutamine residue, we analyzed all possible substitutions of this residue in multiple Gα isoforms. Through cell-based measurements of activity, we showed that some mutants were further activated and inactivated by G protein-coupled receptors. Through biochemical, molecular dynamics, and nuclear magnetic resonance-based structural studies, we showed that the Gα mutants were functionally distinct and conformationally diverse, despite their shared inability to hydrolyze GTP. Thus, the catalytic glutamine residue contributes to functions beyond GTP hydrolysis, and these functions include subtype-specific, allosteric modulation of receptor-mediated subunit dissociation. We conclude that G proteins do not function as simple on-off switches. Rather, signaling emerges from an ensemble of active states, a subset of which are favored in disease and may be uniquely responsive to receptor-directed ligands.

Metabolic acidosis is undertreated and underdiagnosed: a retrospective cohort study.

BACKGROUND: Guidelines recommend treatment of metabolic acidosis (MA) in patients with chronic kidney disease (CKD), but the diagnosis and treatment rates in real-world settings are unknown. We investigated the frequency of MA treatment and diagnosis in patients with CKD. METHODS: In this retrospective cohort study, we examined administrative health data from two US databases [Optum's de-identified Integrated Claims + Clinical Electronic Health Record Database (US EMR cohort; 1 January 2007 to 30 June 2019) and Symphony Health Solutions IDV® (US claims cohort; 1 May 2016 to 30 April 2019)] and population-level databases from Manitoba, Canada (1 April 2006 to 31 March 2018). Patients who met laboratory criteria indicative of CKD and chronic MA were included: two consecutive estimated glomerular filtration results <60 mL/min/1.73 m2 and two serum bicarbonate results 12 to <22 mEq/L over 28-365 days. Outcomes included treatment of MA (defined as a prescription for oral sodium bicarbonate) and a diagnosis of MA (defined using administrative records). Outcomes were assessed over a 3-year period (1 year pre-index, 2 years post-index). RESULTS: A total of 96 184 patients were included: US EMR, 6179; Manitoba, 3223; US Claims, 86 782. Sodium bicarbonate treatment was prescribed for 17.6%, 8.7% and 15.3% of patients, and a diagnosis was found for 44.7%, 20.9% and 20.9% of patients, for the US EMR, Manitoba and US Claims cohorts, respectively. CONCLUSIONS: This analysis of 96 184 patients with laboratory-confirmed MA from three independent cohorts of patients with CKD and MA highlights an important diagnosis and treatment gap for this disease-modifying complication.

Introducing ethanol ablation as a treatment for thyroid cysts in the emergency setting.

BACKGROUND: Ethanol ablation for the treatment of thyroid cysts has been well documented in the literature as a safe, effective treatment option in the elective setting. This study demonstrates the use of ethanol ablation in the emergency setting. METHODS: Three patients presenting with airway-threatening compressive symptoms secondary to a thyroid cyst were treated with ethanol ablation within 24 hours of presentation to hospital. RESULTS: All patients had symptom resolution at a median of nine months follow up post procedure. Sixty-six per cent of patients required only one treatment. There was a median of 100 per cent radiological resolution of the cystic component. The median Glasgow Benefit Inventory score was +27.7, similar to that for tonsillectomy. CONCLUSION: Ethanol ablation is a safe, cost-effective and efficient treatment option for thyroid cysts in the acute setting.

Surface Plasmon Resonance Screening to Identify Active and Selective Adenosine Receptor Binding Fragments.

Surface plasmon resonance (SPR) is a widely used method to study ligand-protein interactions. The throughput and sensitivity of SPR has made it an important technology for measuring low-affinity, ultralow weight fragments (<200 Da) in the early stages of drug discovery. However, the biochemistry of membrane proteins, such as G-protein-coupled receptors (GPCRs), makes their SPR fragment screening particularly challenging, especially for native/wild-type, nonthermostabilized mutant receptors. In this study, we demonstrate the use of SPR-based biosensors to study the entire human family of adenosine receptors and present biologically active novel binders with a range of selectivity to human adenosine 2a receptor (hA2AR) from an ultralow weight fragment library and the public GlaxoSmithKline (GSK) kinase library. Thus, we demonstrate the ability of SPR to screen ultra-low-affinity fragments and identify biologically meaningful chemical equity and that SPR campaigns are highly effective "chemical filters" for screening small building block fragments that can be used to enable drug discovery programs.

Clinical, radiologic, and pathologic features and outcomes of pulmonary transthyretin amyloidosis.

INTRODUCTION: Amyloid transthyretin amyloidosis (ATTR) is characterized by deposition of a misfolded conformation of the transport protein TTR, most commonly in cardiac and nerve tissue, causing clinical disease. Pulmonary amyloidosis, or deposition of ATTR in lung tissue, is a poorly characterized manifestation of this disease. We present the clinical course, imaging characteristics, pathology results, and outcomes of a patient cohort diagnosed with pulmonary ATTR. METHODS: We retrospectively reviewed records of 28 patients with pulmonary ATTR seen at Mayo Clinic from September 30, 2005, through December 31, 2020. Data collected included information on demographics, subjective symptoms, tissue biopsy results, pulmonary function testing, imaging findings, and treatment. RESULTS: Of the patients, 89% were men; the median age was 74.5 years (range, 50-99 years). Patients were typically diagnosed after persistent dyspnea and abnormal chest imaging resulted in lung biopsy, which yielded the ATTR diagnosis. Most patients had a preexisting diagnosis of cardiac ATTR. The disease was wild-type in 62% and hereditary in 38%. Normal pulmonary function tests followed by a restrictive pattern were the most common presentation. Of the patients, 93% had chest computed tomography, with common findings of diffuse nodularity, calcified granulomas, interlobular septal thickening, and pleural effusions. Almost all patients had pulmonary vascular involvement, and half had interstitial involvement on tissue biopsy. One-third received either anti-amyloid pharmacotherapy or a heart transplant. Half of patients had died before the time of study inclusion. CONCLUSION: Pulmonary disease is a less common but clinically important manifestation of ATTR.

Nanobody-enabled monitoring of kappa opioid receptor states.

Recent studies show that GPCRs rapidly interconvert between multiple states although our ability to interrogate, monitor and visualize them is limited by a relative lack of suitable tools. We previously reported two nanobodies (Nb39 and Nb6) that stabilize distinct ligand- and efficacy-delimited conformations of the kappa opioid receptor. Here, we demonstrate via X-ray crystallography a nanobody-targeted allosteric binding site by which Nb6 stabilizes a ligand-dependent inactive state. As Nb39 stabilizes an active-like state, we show how these two state-dependent nanobodies can provide real-time reporting of ligand stabilized states in cells in situ. Significantly, we demonstrate that chimeric GPCRs can be created with engineered nanobody binding sites to report ligand-stabilized states. Our results provide both insights regarding potential mechanisms for allosterically modulating KOR with nanobodies and a tool for reporting the real-time, in situ dynamic range of GPCR activity.

XFEL structures of the human MT2 melatonin receptor reveal the basis of subtype selectivity.

The human MT1 and MT2 melatonin receptors1,2 are G-protein-coupled receptors (GPCRs) that help to regulate circadian rhythm and sleep patterns3. Drug development efforts have targeted both receptors for the treatment of insomnia, circadian rhythm and mood disorders, and cancer3, and MT2 has also been implicated in type 2 diabetes4,5. Here we report X-ray free electron laser (XFEL) structures of the human MT2 receptor in complex with the agonists 2-phenylmelatonin (2-PMT) and ramelteon6 at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants: H2085.46A (superscripts represent the Ballesteros-Weinstein residue numbering nomenclature7) and N862.50D, obtained in complex with 2-PMT. Comparison of the structures of MT2 with a published structure8 of MT1 reveals that, despite conservation of the orthosteric ligand-binding site residues, there are notable conformational variations as well as differences in [3H]melatonin dissociation kinetics that provide insights into the selectivity between melatonin receptor subtypes. A membrane-buried lateral ligand entry channel is observed in both MT1 and MT2, but in addition the MT2 structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data that support a prominent role for intramembrane ligand entry in both receptors, and suggest that there might also be an extracellular entry path in MT2. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents.

Structural basis of ligand recognition at the human MT1 melatonin receptor.

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.

Elective Naloxone-Induced Opioid Withdrawal for Rapid Initiation of Medication-Assisted Treatment of Opioid Use Disorder.

We present a case of elective naloxone-induced opioid withdrawal followed by buprenorphine rescue to initiate opioid use disorder treatment in the emergency department. This strategy may represent a safe alternative to prescribing buprenorphine for outpatient initiation, a method that puts the patient at risk for complications of unmonitored opioid withdrawal, including relapse. After confirmation that the naloxone-induced withdrawal was adequately treated with buprenorphine, the patient was discharged with prescribed buprenorphine to follow up in an addiction medicine clinic, where he was treated 2 days later.

The Fracture Risk Assessment Tool (FRAX®) predicts fracture risk in patients with chronic kidney disease.

The Fracture Risk Assessment Tool (FRAX®) was developed to predict fracture risk in the general population, but its applicability to patients with chronic kidney disease (CKD) is unknown. Using the Manitoba Bone Mineral Density (BMD) Database, we identified adults not receiving dialysis with available serum creatinine measurements and bone densitometry within 1 year. Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation. Incident major osteoporotic fractures and hip fractures were ascertained from population-based health care databases. The performance of FRAX, derived without and with BMD, was studied in relation to CKD stage. Among 10,099 subjects (mean age 64 ± 13 years, 13.0% male), 2,154 had eGFR 30-60 mL/min/1.73 m2 (CKD stage 3) and 590 had eGFR <30 mL/min/1.73 m2 (CKD stages 4-5). During a 5-year observation period, 772 individuals experienced a major osteoporotic fracture and 226 had a hip fracture. FRAX predicted risk for major osteoporotic fracture and hip fracture in all eGFR strata. For every standard deviation increase in FRAX score derived with BMD, the hazard ratio (HR) for hip fracture was 4.54 (95% confidence interval [CI] 3.57-5.77) in individuals with eGFR ≥ 60 mL/min/1.73m2, 4.52 (95% CI 3.15-6.49) in individuals with eGFR 30-60 mL/min/1.73m2, and 3.10 (95% CI 1.80-5.33) in individuals with eGFR <30 mL/min/1.73m2. The relationship between FRAX and major osteoporotic fracture was stronger in those with CKD compared to those with preserved eGFR. These findings support the use of FRAX to risk stratify patients with non-dialysis CKD for major osteoporotic fractures and hip fractures.

Space Radiation Alters Genotype-Phenotype Correlations in Fear Learning and Memory Tests.

Behavioral and cognitive traits have a genetic component even though contributions from individual genes and genomic loci are in many cases modest. Changes in the environment can alter genotype-phenotype relationships. Space travel, which includes exposure to ionizing radiation, constitutes environmental challenges and is expected to induce not only dramatic behavioral and cognitive changes but also has the potential to induce physical DNA damage. In this study, we utilized a genetically heterogeneous mouse model, dense genotype data, and shifting environmental challenges, including ionizing radiation exposure, to explore and quantify the size and stability of the genetic component of fear learning and memory-related measures. Exposure to ionizing radiation and other external stressors altered the genotype-phenotype correlations, although different behavioral and cognitive measures were affected to different extents. Utilizing an integrative genomic approach, we identified pathways and functional ontology categories associated with these behavioral and cognitive measures.

Trim9 Deletion Alters the Morphogenesis of Developing and Adult-Born Hippocampal Neurons and Impairs Spatial Learning and Memory.

UNLABELLED: During hippocampal development, newly born neurons migrate to appropriate destinations, extend axons, and ramify dendritic arbors to establish functional circuitry. These developmental stages are recapitulated in the dentate gyrus of the adult hippocampus, where neurons are continuously generated and subsequently incorporate into existing, local circuitry. Here we demonstrate that the E3 ubiquitin ligase TRIM9 regulates these developmental stages in embryonic and adult-born mouse hippocampal neurons in vitro and in vivo Embryonic hippocampal and adult-born dentate granule neurons lacking Trim9 exhibit several morphological defects, including excessive dendritic arborization. Although gross anatomy of the hippocampus was not detectably altered by Trim9 deletion, a significant number of Trim9(-/-) adult-born dentate neurons localized inappropriately. These morphological and localization defects of hippocampal neurons in Trim9(-/-) mice were associated with extreme deficits in spatial learning and memory, suggesting that TRIM9-directed neuronal morphogenesis may be involved in hippocampal-dependent behaviors. SIGNIFICANCE STATEMENT: Appropriate generation and incorporation of adult-born neurons in the dentate gyrus are critical for spatial learning and memory and other hippocampal functions. Here we identify the brain-enriched E3 ubiquitin ligase TRIM9 as a novel regulator of embryonic and adult hippocampal neuron shape acquisition and hippocampal-dependent behaviors. Genetic deletion of Trim9 elevated dendritic arborization of hippocampal neurons in vitro and in vivo Adult-born dentate granule cells lacking Trim9 similarly exhibited excessive dendritic arborization and mislocalization of cell bodies in vivo These cellular defects were associated with severe deficits in spatial learning and memory.

Cre-dependent DREADD (Designer Receptors Exclusively Activated by Designer Drugs) mice.

DREADDs, designer receptors exclusively activated by designer drugs, are engineered G protein-coupled receptors (GPCR) which can precisely control GPCR signaling pathways (for example, Gq, Gs, and Gi). This chemogenetic technology for control of GPCR signaling has been successfully applied in a variety of in vivo studies, including in mice, to remotely control GPCR signaling, for example, in neurons, glia cells, pancreatic ß-cells, or cancer cells. In order to fully explore the in vivo applications of the DREADD technology, we generated hM3Dq and hM4Di strains of mice which allow for Cre recombinase-mediated restricted expression of these pathway-selective DREADDs. With the many Cre driver lines now available, these DREADD lines will be applicable to studying a wide array of research and preclinical questions. genesis 54:439-446, 2016. © 2016 Wiley Periodicals, Inc.

Effects of Proton and Combined Proton and (56)Fe Radiation on the Hippocampus.

The space radiation environment contains protons and (56)Fe, which could pose a significant hazard to space flight crews during and after missions. The space environment involves complex radiation exposures, thus, the effects of a dose of protons might be modulated by a dose of heavy-ion radiation. The brain, and particularly the hippocampus, may be susceptible to space radiation-induced changes. In this study, we first determined the dose-response effect of proton radiation (150 MeV) on hippocampus-dependent cognition 1 and 3 months after exposure. Based on those results, we subsequently exposed mice to protons alone (150 MeV, 0.1 Gy), (56)Fe alone (600 MeV/n, 0.5 Gy) or combined proton and (56)Fe radiations (protons first) with the two exposures separated by 24 h. At one month postirradiation, all animal groups showed novel object recognition. However, at three months postirradiation, mice exposed to either protons or combined proton and (56)Fe radiations showed impaired novel object recognition, which was not observed in mice irradiated with (56)Fe alone. The mechanisms in these impairments might involve inflammation. In mice irradiated with protons alone or (56)Fe alone three months earlier, there was a negative correlation between a measure of novel object recognition and the number of newly born activated microglia in the dentate gyrus. Next, cytokine and chemokine levels were assessed in the hippocampus. At one month after exposure the levels of IL-12 were higher in mice exposed to combined radiations compared with sham-irradiated mice, while the levels of IFN-γ were lower in mice exposed to (56)Fe radiation alone or combined radiations. In addition, IL-4 levels were lower in (56)Fe-irradiated mice compared with proton-irradiated mice and TNF-α levels were lower in proton-irradiated mice than in mice receiving combined radiations. At three months after exposure, macrophage-derived chemokine (MDC) and eotaxin levels were lower in mice receiving combined radiations. The levels of MDC and eotaxin correlated and the levels of MDC, but not eotaxin, correlated with the percentage of newly born activated microglia in the blades of the dentate gyrus. Finally, hippocampal IL-6 levels were higher in mice receiving combined radiations compared with mice receiving (56)Fe radiation alone. These data demonstrate the sensitivity of novel object recognition for detecting cognitive injury three months after exposure to proton radiation alone, and combined exposure to proton and (56)Fe radiations, and that newly-born activated microglia and inflammation might be involved in this injury.

Combined exposure to protons and (56)Fe leads to overexpression of Il13 and reactivation of repetitive elements in the mouse lung.

Interest in deep space exploration underlines the needs to investigate the effects of exposure to combined sources of space radiation. The lung is a target organ for radiation, and exposure to protons and heavy ions as radiation sources may lead to the development of degenerative disease and cancer. In this study, we evaluated the pro-fibrotic and epigenetic effects of exposure to protons (150 MeV/nucleon, 0.1 Gy) and heavy iron ions ((56)Fe, 600 MeV/nucleon, 0.5 Gy) alone or in combination (protons on Day 1 and (56)Fe on Day 2) in C57BL/6 male mice 4 weeks after irradiation. Exposure to (56)Fe, proton or in combination, did not result in histopathological changes in the murine lung. At the same time, combined exposure to protons and (56)Fe resulted in pronounced molecular alterations in comparison with either source of radiation alone. Specifically, we observed a substantial increase in the expression of cytokine Il13, loss of expression of DNA methyltransferase Dnmt1, and reactivation of LINE-1, SINE B1 retrotransposons, and major and minor satellites. Given the deleterious potential of the observed effects that may lead to development of chronic lung injury, pulmonary fibrosis, and cancer, future studies devoted to the investigation of the long-term effects of combined exposures to proton and heavy ions are clearly needed.

Impairment in extinction of contextual and cued fear following post-training whole-body irradiation.

Because of the use of radiation in cancer therapy, the risk of nuclear contamination from power plants, military conflicts, and terrorism, there is a compelling scientific and public health interest in the effects of environmental radiation exposure on brain function, in particular hippocampal function and learning and memory. Previous studies have emphasized changes in learning and memory following radiation exposure. These approaches have ignored the question of how radiation exposure might impact recently acquired memories, which might be acquired under traumatic circumstances (cancer treatment, nuclear disaster, etc.). To address the question of how radiation exposure might affect the processing and recall of recently acquired memories, we employed a fear conditioning paradigm wherein animals were trained, and subsequently irradiated (whole-body X-ray irradiation) 24 h later. Animals were given 2 weeks to recover, and were tested for retention and extinction of hippocampus-dependent contextual fear conditioning or hippocampus-independent cued fear conditioning. Exposure to irradiation following training was associated with reduced daily increases in body weights over the 22-days of the study and resulted in greater freezing levels and aberrant extinction 2 weeks later. This was also observed when the intensity of the training protocol was increased. Cued freezing levels and measures of anxiety 2 weeks after training were also higher in irradiated than sham-irradiated mice. In contrast to contextual freezing levels, cued freezing levels were even higher in irradiated mice receiving 5 shocks during training than sham-irradiated mice receiving 10 shocks during training. In addition, the effects of radiation on extinction of contextual fear were more profound than those on the extinction of cued fear. Thus, whole-body irradiation elevates contextual and cued fear memory recall.

Pathogenesis of scrapie in ARQ/ARQ sheep after subcutaneous infection: effect of lymphadenectomy and immune cell subset changes in relation to prion protein accumulation.

It is well established that the infectious agent of scrapie can replicate in the lymphoreticular system (LRS). However, the effects of removal of LRS target tissues on the pathogenesis of the infection and the accumulation of disease-associated prion protein (PrP(d)) in LRS tissues on specific immune cell subsets are poorly understood aspects. To address these questions 16 ARQ/ARQ sheep were subcutaneously inoculated in the drainage area of the prefemoral lymph node with brain homogenate derived from Suffolk sheep naturally infected with scrapie. Fourteen sheep were then subjected to either early (14-17 days post-inoculation [dpi]) or late (175-201 dpi) lymphadenectomy and culled at preclinical or clinical stages of infection. Neither late nor even early lymphadenectomy prevented infection or had any effect on the accumulation of PrP(d) in the LRS or CNS suggesting a rapid organic dissemination of the infectious agent after inoculation. Lymph nodes from eight scrapie inoculated sheep selected on the basis of the amount of PrP(d) in their LRS tissues (negative, low or high) were examined for six different immune cell markers. The PrP(d) negative lymph nodes from two sheep with no evidence of scrapie infection showed lower numbers of cluster of determination (CD) 21 positive cells than PrP(d) positive nodes, irrespective of their location (hind leg or head). However, quantitative differences in the expression of this marker were not detected when comparing lymph nodes with low and high levels of PrP(d) accumulation, suggesting that proliferation of CD21 positive cells is related to scrapie infection, but not directly linked to the magnitude of PrP(d) accumulation. An additional observation of the study was that sheep that were methionin-threonine at codon 112 of the prion protein gene showed lower attack rates than methionine homozygotes (67% and 100%, respectively) and also generally lower levels of PrP(d) accumulation in the LRS and brain and increased survival times, suggesting an influence of such polymorphism in the susceptibility to scrapie.

Neuropathological changes correlate temporally but not spatially with selected neuromodulatory responses in natural scrapie.

AIM: Neuropathological changes classically associated with sheep scrapie do not always correlate with clinical disease. We aimed to determine if selected neuromodulatory responses were altered during the course of the infection as it has been described in Creutzfeldt-Jakob disease and experimental bovine spongiform encephalopathy. METHODS: Hemi-brains from healthy sheep and natural scrapie cases at two stages of infection were examined for biochemical alterations related to the expression of type I metabotropic glutamatergic receptors (mGluR(1) ) and type I adenosine receptors I (A(1) R), and of selected downstream intermediate signalling targets. Immunohistochemistry for different scrapie-related neuropathological changes was performed in the contralateral hemi-brains. RESULTS: PrP(d) deposition, spongiform change, astrocytosis and parvalbumin expression were significantly altered in brains from clinically affected sheep compared with preclinical cases and negative controls; the latter also showed significantly higher immunoreactivity for synaptophysin than clinical cases. Between clinically affected and healthy sheep, no differences were found in the protein levels of mGluR(1) , while phospholipase Cß1 expression in terminally ill sheep was increased in some brain areas but decreased in others. Adenyl cyclase 1 and A(1) R levels were significantly lower in various brain areas of affected sheep. No abnormal biochemical expression levels of these markers were found in preclinically infected sheep. CONCLUSIONS: These findings point towards an involvement of mGluR(1) and A(1) R downstream pathways in natural scrapie. While classical prion disease lesions and neuromodulatory responses converge in some affected regions, they do not do so in others suggesting that there are independent regulatory factors for distinct degenerative and neuroprotective responses.