A New Minimal-Stress Freely-Moving Rat Model for Preclinical Studies on Intranasal Administration of CNS Drugs

Purpose. To develop a new minimal-stress model for intranasal administration in freely moving rats and to evaluate in this model the brain distribution of acetaminophen following intranasal versus intravenous administration. Methods. Male Wistar rats received one intranasal cannula, an intra-cerebral microdialysis probe, and two blood cannulas for drug administration and serial blood sampling respectively. To evaluate this novel model, the following experiments were conducted. 1) Evans Blue was administered to verify the selectivity of intranasal exposure. 2) During a 1 min infusion 10, 20, or 40 μl saline was administered intranasally or 250 µl intravenously. Corticosterone plasma concentrations over time were compared as biomarkers for stress. 3) 200 µg of the model drug acetaminophen was given in identical setup and plasma, and brain pharmacokinetics were determined. Results. In 96 % of the rats, only the targeted nasal cavity was deeply colored. Corticosterone plasma concentrations were not influenced, neither by route nor volume of administration. Pharmacokinetics of acetaminophen were identical after intravenous and intranasal administration, although the Cmax in microdialysates was reached a little earlier following intravenous administration. Conclusion. A new minimal-stress model for intranasal administration in freely moving rats has been successfully developed and allows direct comparison with intravenous administration. KEY WORDS: acetaminophen; brain; intranasal infusion; microdialysis; pharmacokinetics

Wolbachia in the flesh: symbiont intensities in germ-line and somatic tissues challenge the conventional view of Wolbachia transmission routes

Symbionts can substantially affect the evolution and ecology of their hosts. The investigation of the tissue-specific distribution of symbionts (tissue tropism) can provide important insight into host-symbiont interactions. Among other things, it can help to discern the importance of specific transmission routes and potential phenotypic effects. The intracellular bacterial symbiont Wolbachia has been described as the greatest ever panzootic, due to the wide array of arthropods that it infects. Being primarily vertically transmitted, it is expected that the transmission of Wolbachia would be enhanced by focusing infection in the reproductive tissues. In social insect hosts, this tropism would logically extend to reproductive rather than sterile castes, since the latter constitute a dead-end for vertically transmission. Here, we show that Wolbachia are not focused on reproductive tissues of eusocial insects, and that non-reproductive tissues of queens and workers of the ant Acromyrmex echinatior, harbour substantial infections. In particular, the comparatively high intensities of Wolbachia in the haemolymph, fat body, and faeces, suggest potential for horizontal transmission via parasitoids and the faecal-oral route, or a role for Wolbachia modulating the immune response of this host. It may be that somatic tissues and castes are not the evolutionary dead-end for Wolbachia that is commonly thought

Early symptoms in symptomatic and preclinical genetic frontotemporal lobar degeneration

OBJECTIVES: The clinical heterogeneity of frontotemporal dementia (FTD) complicates identification of biomarkers for clinical trials that may be sensitive during the prediagnostic stage. It is not known whether cognitive or behavioural changes during the preclinical period are predictive of genetic status or conversion to clinical FTD. The first objective was to evaluate the most frequent initial symptoms in patients with genetic FTD. The second objective was to evaluate whether preclinical mutation carriers demonstrate unique FTD-related symptoms relative to familial mutation non-carriers. METHODS: The current study used data from the Genetic Frontotemporal Dementia Initiative multicentre cohort study collected between 2012 and 2018. Participants included symptomatic carriers (n=185) of a pathogenic mutation in chromosome 9 open reading frame 72 (C9orf72), progranulin (GRN) or microtubule-associated protein tau (MAPT) and their first-degree biological family members (n=588). Symptom endorsement was documented using informant and clinician-rated scales. RESULTS: The most frequently endorsed initial symptoms among symptomatic patients were apathy (23%), disinhibition (18%), memory impairments (12%), decreased fluency (8%) and impaired articulation (5%). Predominant first symptoms were usually discordant between family members. Relative to biologically related non-carriers, preclinical MAPT carriers endorsed worse mood and sleep symptoms, and C9orf72 carriers endorsed marginally greater abnormal behaviours. Preclinical GRN carriers endorsed less mood symptoms compared with non-carriers, and worse everyday skills. CONCLUSION: Preclinical mutation carriers exhibited neuropsychiatric symptoms compared with non-carriers that may be considered as future clinical trial outcomes. Given the heterogeneity in symptoms, the detection of clinical transition to symptomatic FTD may be best captured by composite indices integrating the most common initial symptoms for each genetic group

Different rates of cognitive decline in autosomal dominant and late-onset Alzheimer disease

As prevention trials advance with autosomal dominant Alzheimer disease (ADAD) participants, understanding the similarities and differences between ADAD and "sporadic" late-onset AD (LOAD) is critical to determine generalizability of findings between these cohorts. Cognitive trajectories of ADAD mutation carriers (MCs) and autopsy-confirmed LOAD individuals were compared to address this question. Longitudinal rates of change on cognitive measures were compared in ADAD MCs (n = 310) and autopsy-confirmed LOAD participants (n = 163) before and after symptom onset (estimated/observed). LOAD participants declined more rapidly in the presymptomatic (preclinical) period and performed more poorly at symptom onset than ADAD participants on a cognitive composite. After symptom onset, however, the younger ADAD MCs declined more rapidly. The similar but not identical cognitive trajectories (declining but at different rates) for ADAD and LOAD suggest common AD pathologies but with some differences

Chronic Intranasal Treatment with an Anti-Aβ30-42 scFv Antibody Ameliorates Amyloid Pathology in a Transgenic Mouse Model of Alzheimer's Disease

Amyloid-beta peptide (Aβ)-directed active and passive immunization therapeutic strategies reduce brain levels of Aβ, decrease the severity of beta-amyloid plaque pathology and reverse cognitive deficits in mouse models of Alzheimer's disease (AD). As an alternative approach to passive immunization with full IgG molecules, single-chain variable fragment (scFv) antibodies can modulate or neutralize Aβ-related neurotoxicity and inhibit its aggregation in vitro. In this study, we characterized a scFv derived from a full IgG antibody raised against the C-terminus of Aβ, and studied its passage into the brains of APP transgenic mice, as well as its potential to reduce Aβ-related pathology. We found that the scFv entered the brain after intranasal application, and that it bound to beta-amyloid plaques in the cortex and hippocampus of APP transgenic mice. Moreover, the scFv inhibited Aβ fibril formation and Aβ-mediated neurotoxicity in vitro. In a preventative therapeutic approach chronic intranasal treatment with scFv reduced congophilic amyloid angiopathy (CAA) and beta-amyloid plaque numbers in the cortex of APPswe/PS1dE9 mice. This reduction of CAA and plaque pathology was associated with a redistribution of brain Aβ from the insoluble fraction to the soluble peptide pool. Due to their lack of the effector domain of full IgG, scFv may represent an alternative tool for the treatment of Aβ-related pathology without triggering Fc-mediated effector functions. Additionally, our observations support the possibility that Aβ-directed immunotherapy can reduce Aβ deposition in brain vessels in transgenic mice

Variant-dependent heterogeneity in amyloid β burden in autosomal dominant Alzheimer's disease: cross-sectional and longitudinal analyses of an observational study

Background: Insights gained from studying individuals with autosomal dominant Alzheimer's disease have broadly influenced mechanistic hypotheses, biomarker development, and clinical trials in both sporadic and dominantly inherited Alzheimer's disease. Although pathogenic variants causing autosomal dominant Alzheimer's disease are highly penetrant, there is substantial heterogeneity in levels of amyloid β (Aβ) between individuals. We aimed to examine whether this heterogeneity is related to disease progression and to investigate the association with mutation location within PSEN1, PSEN2, or APP. Methods: We did cross-sectional and longitudinal analyses of data from the Dominantly Inherited Alzheimer's Network (DIAN) observational study, which enrols individuals from families affected by autosomal dominant Alzheimer's disease. 340 participants in the DIAN study who were aged 18 years or older, had a history of autosomal dominant Alzheimer's disease in their family, and who were enrolled between September, 2008, and June, 2019, were included in our analysis. 206 participants were carriers of pathogenic mutations in PSEN1, PSEN2, or APP, and 134 were non-carriers. 62 unique pathogenic variants were identified in the cohort and were grouped in two ways. First, we sorted variants in PSEN1, PSEN2, or APP by the affected protein domain. Second, we divided PSEN1 variants according to position before or after codon 200. We examined variant-dependent variability in Aβ biomarkers, specifically Pittsburgh-Compound-B PET (PiB-PET) signal, levels of CSF Aβ1-42 (Aβ42), and levels of Aβ1-40 (Aβ40). Findings: Cortical and striatal PiB-PET signal showed striking variant-dependent variability using both grouping approaches (p0·7), and CSF Aβ42 levels (codon-based grouping: p=0·49; domain-based grouping: p=0·095). Longitudinal PiB-PET signal also varied across codon-based groups, mirroring cross-sectional analyses. Interpretation: Autosomal dominant Alzheimer's disease pathogenic variants showed highly differential temporal and regional patterns of PiB-PET signal, despite similar functional progression. These findings suggest that although increased PiB-PET signal is generally seen in autosomal dominant Alzheimer's disease, higher levels of PiB-PET signal at an individual level might not reflect more severe or more advanced disease. Our results have high relevance for ongoing clinical trials in autosomal dominant Alzheimer's disease, including those using Aβ PET as a surrogate marker of disease progression. Additionally, and pertinent to both sporadic and autosomal dominant Alzheimer's disease, our results suggest that CSF and PET measures of Aβ levels are not interchangeable and might reflect different Aβ-driven pathobiological processes. Funding: National Institute on Aging, Doris Duke Charitable Foundation, German Center for Neurodegenerative Diseases, Japanese Agency for Medical Research and Development

Genetically-controlled Vesicle-Associated Membrane Protein 1 expression may contribute to Alzheimer’s pathophysiology and susceptibility

Background Alzheimer’s disease is a neurodegenerative disorder in which extracellular deposition of β-amyloid (Aβ) oligomers causes synaptic injury resulting in early memory loss, altered homeostasis, accumulation of hyperphosphorylated tau and cell death. Since proteins in the SNAP (Soluble N-ethylmaleimide-sensitive factor Attachment Protein) REceptors (SNARE) complex are essential for neuronal Aβ release at pre-synaptic terminals, we hypothesized that genetically controlled SNARE expression could alter neuronal Aß release at the synapse and hence play an early role in Alzheimer’s pathophysiology. Results Here we report 5 polymorphisms in Vesicle-Associated Membrane Protein 1 (VAMP1), a gene encoding a member of the SNARE complex, associated with bidirectionally altered cerebellar VAMP1 transcript levels (all p < 0.05). At the functional level, we demonstrated that control of VAMP1 expression by heterogeneous knockdown in mice resulted in up to 74% reduction in neuronal Aβ exocytosis (p < 0.001). We performed a case-control association study of the 5 VAMP1 expression regulating polymorphisms in 4,667 Alzheimer’s disease patients and 6,175 controls to determine their contribution to Alzheimer’s disease risk. We found that polymorphisms associated with increased brain VAMP1 transcript levels conferred higher risk for Alzheimer’s disease than those associated with lower VAMP1 transcript levels (p = 0.03). Moreover, we also report a modest protective association for a common VAMP1 polymorphism with Alzheimer’s disease risk (OR = 0.88, p = 0.03). This polymorphism was associated with decreased VAMP1 transcript levels (p = 0.02) and was functionally active in a dual luciferase reporter gene assay (p < 0.01). Conclusions Genetically regulated VAMP1 expression in the brain may modify both Alzheimer’s disease risk and may contribute to Alzheimer’s pathophysiology

Co-culturing human prostate carcinoma cells with hepatocytes leads to increased expression of E-cadherin

Metastasis is a multi-step process wherein tumour cells detach from the primary mass, migrate through barrier matrices, gain access to conduits to disseminate, and subsequently survive and proliferate in an ectopic site. During the initial invasion stage, prostate carcinoma cells undergo epithelial–mesenchymal-like transition with gain of autocrine signalling and loss of E-cadherin, hallmarks that appear to enable invasion and dissemination. However, some metastases express E-cadherin, and we found close connections between prostate carcinoma cells and hepatocytes in a liver microtissue bioreactor. We hypothesise that phenotypic plasticity occurs late in prostate cancer progression at the site of ectopic seeding. Immunofluorescence staining for E-cadherin in co-cultures of hepatocytes and DU-145 prostate cancer cells revealed E-cadherin upregulation at peripheral sites of contact by day 2 of co-culture; E-cadherin expression also increased in PC-3 cells in co-culture. These carcinoma cells bound to hepatocytes in an E-cadherin-dependent manner. Although the signals by which the hepatocytes elicited E-cadherin expression remain undetermined, it appeared related to downregulation of epidermal growth factor receptor (EGFR) signalling. Inhibition of autocrine EGFR signalling increased E-cadherin expression and cell–cell heterotypic adhesion; further, expression of a downregulation-resistant EGFR variant prevented E-cadherin upregulation. These findings were supported by finding E-cadherin and catenins but not activated EGFR in human prostate metastases to the liver. We conclude that the term epithelial–mesenchymal transition only summarises the transient downregulation of E-cadherin for invasion with re-expression of E-cadherin being a physiological consequence of metastatic seeding

Molecular pathways involved in the synergistic interaction of the PKCβ inhibitor enzastaurin with the antifolate pemetrexed in non-small cell lung cancer cells

Conventional regimens have limited impact against non-small cell lung cancer (NSCLC). Current research is focusing on multiple pathways as potential targets, and this study investigated molecular mechanisms underlying the combination of the PKCβ inhibitor enzastaurin with the multitargeted antifolate pemetrexed in the NSCLC cells SW1573 and A549. Pharmacologic interaction was studied using the combination-index method, while cell cycle, apoptosis induction, VEGF secretion and ERK1/2 and Akt phosphorylation were studied by flow cytometry and ELISAs. Reverse transcription–PCR, western blot and activity assays were performed to assess whether enzastaurin influenced thymidylate synthase (TS) and the expression of multiple targets involved in cancer signaling and cell cycle distribution. Enzastaurin-pemetrexed combination was highly synergistic and significantly increased apoptosis. Enzastaurin reduced both phosphoCdc25C, resulting in G2/M checkpoint abrogation and apoptosis induction in pemetrexed-damaged cells, and GSK3β and Akt phosphorylation, which was additionally reduced by drug combination (−58% in A549). Enzastaurin also significantly reduced pemetrexed-induced upregulation of TS expression, possibly through E2F-1 reduction, whereas the combination decreased TS in situ activity (>50% in both cell lines) and VEGF secretion. The effects of enzastaurin on signaling pathways involved in cell cycle control, apoptosis and angiogenesis, as well as on the expression of genes involved in pemetrexed activity provide a strong experimental basis to their evaluation as pharmacodynamic markers in clinical trials of enzastaurin-pemetrexed combination in NSCLC patients

Pattern and degree of individual brain atrophy predicts dementia onset in dominantly inherited Alzheimer's disease

Introduction: Asymptomatic and mildly symptomatic dominantly inherited Alzheimer's disease mutation carriers (DIAD-MC) are ideal candidates for preventative treatment trials aimed at delaying or preventing dementia onset. Brain atrophy is an early feature of DIAD-MC and could help predict risk for dementia during trial enrollment. Methods: We created a dementia risk score by entering standardized gray-matter volumes from 231 DIAD-MC into a logistic regression to classify participants with and without dementia. The score's predictive utility was assessed using Cox models and receiver operating curves on a separate group of 65 DIAD-MC followed longitudinally. Results: Our risk score separated asymptomatic versus demented DIAD-MC with 96.4% (standard error = 0.02) and predicted conversion to dementia at next visit (hazard ratio = 1.32, 95% confidence interval [CI: 1.15, 1.49]) and within 2 years (area under the curve = 90.3%, 95% CI [82.3%–98.2%]) and improved prediction beyond established methods based on familial age of onset. Discussion: Individualized risk scores based on brain atrophy could be useful for establishing enrollment criteria and stratifying DIAD-MC participants for prevention trials