Cognitive, physical, and mental health outcomes between long-term cannabis and tobacco users.

INTRODUCTION: Cannabis intoxication adversely affects health, yet persistent effects following short-term abstinence in long-term cannabis users are unclear. This matched-subjects, cross-sectional study compared health outcomes of long-term cannabis and long-term tobacco-only users, relative to population norms. METHODS: Nineteen long-term (mean 32.3years of use, mean age 55.7years), abstinent (mean 15h) cannabis users and 16 long-term tobacco users (mean 37.1years of use, mean age 52.9years), matched for age, educational attainment, and lifetime tobacco consumption, were compared on measures of learning and memory, response inhibition, information-processing, sustained attention, executive control, and mental and physical health. RESULTS: Cannabis users exhibited poorer overall learning and delayed recall and greater interference and forgetting than tobacco users, and exhibited poorer recall than norms. Inhibition and executive control were similar between groups, but cannabis users had slower reaction times during information processing and sustained attention tasks. Cannabis users had superior health satisfaction and psychological, somatic, and general health than tobacco users and had similar mental and physical health to norms whilst tobacco users had greater stress, role limitations from emotional problems, and poorer health satisfaction. CONCLUSIONS: Long-term cannabis users may exhibit deficits in some cognitive domains despite short-term abstinence and may therefore benefit from interventions to improve cognitive performance. Tobacco alone may contribute to adverse mental and physical health outcomes, which requires appropriate control in future studies.

ß-Hairpin mimics containing a piperidine-pyrrolidine scaffold modulate the ß-amyloid aggregation process preserving the monomer species.

Alzheimer's disease is a neurodegenerative disorder linked to oligomerization and fibrillization of amyloid ß peptides, with Aß1-42 being the most aggregative and neurotoxic one. We report herein the synthesis and conformational analysis of Aß1-42-amyloid related ß-hairpin peptidomimetics, built on a piperidine-pyrrolidine semi rigid ß-turn inducer and bearing two small recognition peptide sequences, designed on oligomeric and fibril structures of Aß1-42. According to these peptide sequences, a stable ß-hairpin or a dynamic equilibrium between two possible architectures was observed. These original constructs are able to greatly delay the kinetics of Aß1-42 aggregation process as demonstrated by thioflavin-T fluorescence, and transmission electron microscopy. Capillary electrophoresis indicates their ability to preserve the monomer species, inhibiting the formation of toxic oligomers. Furthermore, compounds protect against toxic effects of Aß on neuroblastoma cells even at substoichiometric concentrations. This study is the first example of acyclic small ß-hairpin mimics possessing such a highly efficient anti-aggregation activity. The protective effect is more pronounced than that observed with molecules which have undergone clinical trials. The structural elements made in this study provide valuable insights in the understanding of the aggregation process and insights to explore the design of novel acyclic ß-hairpin targeting other types of amyloid-forming proteins.

Self-Assembly and Anti-Amyloid Cytotoxicity Activity of Amyloid beta Peptide Derivatives.

The self-assembly of two derivatives of KLVFF, a fragment Aß(16-20) of the amyloid beta (Aß) peptide, is investigated and recovery of viability of neuroblastoma cells exposed to Aß (1-42) is observed at sub-stoichiometric peptide concentrations. Fluorescence assays show that NH2-KLVFF-CONH2 undergoes hydrophobic collapse and amyloid formation at the same critical aggregation concentration (cac). In contrast, NH2-K(Boc)LVFF-CONH2 undergoes hydrophobic collapse at a low concentration, followed by amyloid formation at a higher cac. These findings are supported by the ß-sheet features observed by FTIR. Electrospray ionization mass spectrometry indicates that NH2-K(Boc)LVFF-CONH2 forms a significant population of oligomeric species above the cac. Cryo-TEM, used together with SAXS to determine fibril dimensions, shows that the length and degree of twisting of peptide fibrils seem to be influenced by the net peptide charge. Grazing incidence X-ray scattering from thin peptide films shows features of ß-sheet ordering for both peptides, along with evidence for lamellar ordering of NH2-KLVFF-CONH2. This work provides a comprehensive picture of the aggregation properties of these two KLVFF derivatives and shows their utility, in unaggregated form, in restoring the viability of neuroblastoma cells against Aß-induced toxicity.

Clinical trials of medicinal cannabis for appetite-related symptoms from advanced cancer: a survey of preferences, attitudes and beliefs among patients willing to consider participation.

BACKGROUND: Australian clinical trials are planned to evaluate medicinal cannabis in a range of clinical contexts. AIMS: To explore the preferences, attitudes and beliefs of patients eligible and willing to consider participation in a clinical trial of medicinal cannabis for poor appetite and appetite-related symptoms from advanced cancer. METHODS: A cross-sectional anonymous survey was administered from July to December 2015 online and in eight adult outpatient palliative care and/or cancer services. Respondents were eligible if they were ≥18 years, had advanced cancer and poor appetite/taste problems/weight loss and might consider participating in a medicinal cannabis trial. Survey items focused on medicinal rather than recreational cannabis use and did not specify botanical or pharmaceutical products. Items asked about previous medicinal cannabis use and preferences for delivery route and invited comments and concerns. RESULTS: There were 204 survey respondents, of whom 26 (13%) reported prior medicinal cannabis use. Tablets/capsules were the preferred delivery mode (n = 144, 71%), followed by mouth spray (n = 84, 42%) and vaporiser (n = 83, 41%). Explanations for preferences (n = 134) most commonly cited convenience (n = 66; 49%). A total of 82% (n = 168) of respondents indicated that they had no trial-related concerns, but a small number volunteered concerns about adverse effects (n = 14) or wanted more information/advice (n = 8). Six respondents volunteered a belief that cannabis might cure cancer, while two wanted assurance of efficacy before participating in a trial. CONCLUSION: Justification of modes other than tablets/capsules and variable understanding about cannabis and trials will need addressing in trial-related information to optimise recruitment and ensure that consent is properly informed.

The production of hydrogen peroxide during early-stage protein aggregation: a common pathological mechanism in different neurodegenerative diseases?

By means of an ESR spin-trapping method, we have shown that Abeta (amyloid beta), alpha-synuclein and various toxic forms of the prion protein all appear to generate H2O2 in vitro. A fundamental molecular mechanism underlying the pathogenesis of cell death in several different neurodegenerative diseases could be the direct production of H2O2 during the early stages of protein aggregation.

alpha-Synuclein implicated in Parkinson's disease catalyses the formation of hydrogen peroxide in vitro.

Some rare inherited forms of Parkinson's disease (PD) are due to mutations in the gene encoding a 140-amino acid presynaptic protein called alpha-synuclein. In PD, and some other related disorders such as dementia with Lewy bodies, alpha-synuclein accumulates in the brain in the form of fibrillar aggregates, which are found inside the neuronal cytoplasmic inclusions known as Lewy bodies. By means of an electron spin resonance (ESR) spin trapping method, we show here that solutions of full-length alpha-synuclein, and a synthetic peptide fragment of alpha-synuclein corresponding to residues 61-95 (the so-called non-Abeta component or NAC), both liberate hydroxyl radicals upon incubation in vitro followed by the addition of Fe(II). We did not observe this property for the related beta- and gamma-synucleins, which are not found in Lewy bodies, and are not linked genetically to any neurodegenerative disorder. There is abundant evidence for the involvement of free radicals and oxidative stress in the pathogenesis of nigral damage in PD. Our new data suggest that the fundamental molecular mechanism underlying this pathological process could be the production of hydrogen peroxide by alpha-synuclein.

Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells.

Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The K(i) values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3xK(i)). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFkappaB activation with IC(50) values comparable to their K(i) values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells.

Introduction to Alzheimer's disease.

In 1907, Alois Alzheimer published an account (1) of a 51-year-old female patient, Auguste D., who suffered from strong feelings of jealousy towards her husband, increased memory impairment, disorientation, hallucinations, and often loud and aggressive behavior. After four and a half years of rapidly deteriorating mental illness, Auguste D died in a completely demented state. Postmortem histological analysis of her brain using the Bielschowsky silver technique revealed dense bundles of unusual fibrils within nerve cells (neurofibrillary tangles or NFTs) and numerous focal lesions within the cerebral cortex, subsequently named "senile plaques" by Simchowicz (2) Fig. 1). This combination of progressive presenile dementia with senile plaques and neurofibrillary tangles came to be known as Alzheimer's disease (AD), a term that was later broadened to include senile forms of dementia with similar neuropathological findings. It was Divry (3) who first demonstrated the presence of amyloid at the center of the senile plaque, by means of Congo red staining. All amyloid deposits were originally thought to be starch-like in nature (hence the name), but it is now apparent that they are formed from a variety of different peptides and proteins (the latest count being 18). All amyloid share the property of a characteristic birefringence under polarized light after staining with Congo red dye, which is due to the presence of well-ordered 10 nm fibrils. The underlying protein component of these fibrils invariably adopts predominantly an antiparallel ß-pleated sheet configuration. Ultrastructural observations have confirmed that the core of the senile plaque consists of large numbers of closely-packed, radiating fibrils, similar in appearance to those seen in other forms of amyloidosis (4,5), and have also revealed the presence of paired helical filaments (PHFs) within the NFTs (6). However, it took more than 50 yr from Divry's original observation to determine the precise chemical nature of the senile plaque amyloid. Many neuropathologists have regarded this amyloid as a "tombstone" (an inert bystander) of AD. However, the advent of molecular genetics has finally and firmly established the central role of amyloid in the pathogenesis of the disease, although this is still disputed by some workers in the field. This introductory chapter is written in support of what has become known as the "amyloid cascade" hypothesis.

Alzheimer's disease: correlation of the suppression of beta-amyloid peptide secretion from cultured cells with inhibition of the chymotrypsin-like activity of the proteasome.

Peptide aldehyde inhibitors of the chymotrypsin-like activity of the proteasome (CLIP) such as N-acetyl-Leu-Leu-Nle-H (or ALLN) have been shown previously to inhibit the secretion of beta-amyloid peptide (A beta) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on A beta formation of a much wider range of peptide-based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide-based inhibitors of beta-amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress A beta formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in A beta formation.

Effect of aluminium on expression and processing of amyloid precursor protein.

The environmental agent aluminium has been extensively investigated for a potential role in the aetiology of Alzheimer's disease. Despite many investigations there is at present no definite proof for any involvement. If aluminium is involved it is possible that its action is mediated through interaction with the synthesis or processing of amyloid precursor protein (APP). The present study compared aluminium loaded IMR-32 neuroblastoma cells and rat brains with control cells and brains to determine if aluminium affected APP expression and/or processing. In the IMR-32 model system aluminium had no effect on steady-state APP mRNA levels or on the ratio of individual isoforms. It also had no quantitative or qualitative effect on APP-immunoreactive bands detected in protein extracts from conditioned medium of these cells. In total cell extracts, aluminium reduced the intensity of APP-immunoreactive bands between 120-105 kDa but had no effect on a 9 kDa band. In rat brains, aluminium had no effect on APP-immunoreactive bands from soluble or insoluble-membranous extracts. The results, in general, provide no evidence for any effect of aluminium on APP expression or processing.

The immunoreactive profile at the N-terminal region of A beta 1-39/40 but not A beta 1-42 changes with transition from monomer/dimer to further peptide aggregates.

Using site-specific antibodies, we assessed the effect of aggregation of various length forms of A beta on the immunoreactive profile of the peptides. All of the antibodies tested reacted with monomeric/dimeric forms of A beta 1-42 and its further aggregates. However, antibodies directed against the 1-24 region of A beta reacted weakly or not at all with A beta 1-39/40 monomers or dimers, but immunoreactivity was enhanced substantially following peptide incubation and aggregation. These results suggest that the conformation of the N-terminal region of monomeric and dimeric A beta 1-39/40 is different from that of aggregated forms, whereas the longer A beta 1-42 does not significantly change its N-terminal conformation during beta-sheet fibril formation. These immunochemical results are consistent with previous structural data, and help to explain the differential effects of A beta 1-39/40 and 1-42 on fibril formation in brain.

Variability of beta-amyloid protein deposited lesions in Down's syndrome brains.

An immunohistochemical study was carried out on the brains of 7 adult Down's syndrome cases (ages 31 to 62) using antibodies to beta-protein, beta-amyloid protein precursor and tau-protein. Variable forms of beta-protein deposited lesions (including senile plaques and cerebrovascular amyloidosis) were observed in extensive areas of the neocortex of all cases and coexistence of both beta-protein amyloid fibrils and beta-amyloid protein precursors was also seen in some of these lesions. Moreover, 3 cases at an advanced stage showed a few plaque-like lesions with beta-protein immunoreactivity in the white matter. The following temporal morphological change is suggested for the pathogenesis of Alzheimer's disease: senile plaque undergo sequential structural changes and beta-protein amyloid deposits in the form of "early plaque" precede the development of tau-immunoreactive neurofibrillary degeneration.

Human IMR-32 neuroblastoma cells as a model cell line in Alzheimer's disease research.

The present study investigated expression and processing of amyloid precursor protein by neuronally differentiated IMR-32 neuroblastoma cells. APP mRNA in these cells was found to consist of approximately 58% APP695, 38% APP751, and < 4% APP770. APP-immunoreactive bands detected in western blots of cellular protein extracts were only detected by anti-APP antibodies to peptides with strong homology to APLP2, suggesting that these bands represent APP-like proteins and not APP itself. This result suggests that previous studies claiming immunodetection of cellular forms of APP may have to be re-evaluated. Four main species of C-terminal truncated, secreted APP were detected in blots of protein extracts from medium conditioned by these cells. The immunoreactive profile of these bands suggested a cleavage site N-terminal to the Lys16-Leu17 bond of alpha-secretase. This, together with differences in number and molecular mass of APP-immunoreactive bands between secreted APP from IMR-32 cells and that from the commonly used PC-12 cells, suggests differences in APP processing between these two neuronally differentiated cell lines. In theory, IMR-32 cells being of human neuronal origin may be a more appropriate cell line to study APP-processing in relation to Alzheimer's disease than the rat phaeochromocytoma PC-12 cell line. Therefore, these detected differences warrant further investigation. Additionally IMR-32 cells under certain tissue culture conditions can form intracellular fibrillary material that reacts with anti-PHF specific antibodies. Neuronally differentiated IMR-32 cells could therefore be used as a model system to investigate possible interactions between APP-processing and PHF formation.

Gerstmann-Sträussler-Scheinker disease showing beta-protein type cerebellar and cerebral amyloid angiopathy.

Cerebral amyloid angiopathy is observed in several brain degenerative disorders, but this pathological condition has received little attention in Gerstmann-Sträussler-Scheinker disease (GSS). We report a 69-year-old man who showed the cardinal features of GSS together with typical and extensive congophilic angiopathy. Immunohistochemical studies revealed that the vast majority of the amyloid plaques present in the brain of this patient were consistently labeled by anti-prion protein (PrP) antibody. Double immunostaining disclosed many additional beta-protein immunoreactive plaque-like lesions, including a special type of "hybrid" plaque with colocalization of PrP and beta-protein (beta-PrP). The vascular amyloid deposits seen in both the cerebellum and cerebrum were immunoreactive only to anti-beta-protein antibody. It seems likely that the extensive deposition of beta-protein amyloid (including brain vascular amyloidosis) seen in this and other similar cases is part of pathology of GSS, although the possibility that this finding is due to ageing or concomitant Alzheimer's disease cannot be completely ruled out.

Endopeptidase 3.4.24.11 converts N-1-(R,S)carboxy-3-phenylpropyl-Ala-Ala-Phe-p-carboxyanilide into a potent inhibitor of angiotensin-converting enzyme.

It was reported recently that N-1-(R,S)carboxy-3-phenylpropyl-Ala-Ala-Phe-p-carboxyanilide (CPP-A-A-F-pAB), an inhibitor of endopeptidase 3.4.24.15 (E-24.15), also inhibits angiotensin-converting enzyme (ACE) from rabbit lung. We have found that this compound is without effect on ACE purified from pig kidney, at a concentration some 1000-fold greater than the Ki reported for inhibition of the enzyme from lung. However, preincubation of CPP-A-A-F-pAB with neutral endopeptidase 3.4.24.11 (E-24.11) does result in potent inhibitory effects on ACE. We have shown this to be due to formation of a fragment, CPP-A-A, the structure of which is closely related to ACE inhibitors such as enalaprilat. CPP-A-A was found to be a potent inhibitor of pig ACE. Under the conditions used it had an IC50 value of 1.6 x 10(-8) M, compared with the value obtained for captopril of 7.5 x 10(-10) M. These results have important implications for studies of E-24.15 when using CPP-A-A-F-pAB in vivo or in crude tissue extracts where E-24.11 might also be present.

Alzheimer amyloid beta/A4 peptide binding sites and a possible 'APP-secretase' activity associated with rat brain cortical membranes.

We carried out ligand binding experiments on membranes from rat brain cortical grey matter using radioiodinated beta/A4 8-17, with non-specific binding determined by the addition of 10 microM unlabelled peptide. Specific, reversible binding amounted to 60-75% of total binding and showed a clear dependence on time, temperature, pH and membrane concentration. Kinetic analyses indicated a high-affinity binding site with an apparent KD of 440 pM. However, the ligand was partly degraded with loss of the Ser8, Lys16 and Leu17 residues. Excision of the two C-terminal amino acids was inhibited by EDTA, EGTA, dithiothreitol or Zn2+ but was stimulated by Ca2+ or Mn2+. These studies demonstrate high-affinity binding sites for beta/A4 8-17 (or its derivatives) in rat brain, suggesting that this region may contain a physiologically important amino acid sequence and identify a potential membrane-associated amyloid precursor protein (APP) secretase activity.

Immunohistochemical and immunoelectron microscopical characterization of cerebrovascular and senile plaque amyloid in aged dogs' brains.

Immunohistochemical and immunoelectron microscopical studies were carried out on 28 aged dogs' brains. Amyloid deposits were seen in the arteries and capillaries in the leptomeninges and in superficial areas of the cortices in 19 (67.9%) of the 28 dogs (10-22 years of age). Immunohistochemically, these amyloid deposits were reactive for anti-beta/A4 antibody. Additionally, a variable number of parenchymal deposits with diffuse beta/A4-immunoreactivity (diffuse plaques) was also noted throughout the cerebral cortex in 24/28 dogs (85.7%). However, these plaque lesions were undetectable in Congo red staining. Electron microscopically, amyloid fibrils, measuring 10 nm in width, were located mainly in the tunica media of the arteries, and in less involved vessels they tended to be present among collagen fibres in the adventitia and smooth muscle cells in the outer layer of the media. The plaque lesions appeared to contain sparse aggregations of amyloid fibrils. In immunoelectron microscopical examinations, all amyloid fibrils in both blood vessels and plaques were selectively labelled by gold particles. These findings indicate that aged dogs can provide a useful experimental model for research into the beta/A4-type of cerebral amyloidosis commonly seen in Alzheimer's disease.