Modelling Amyloidosis in Mice

Amyloidosis is a group of disorders in which specific soluble proteins convert into insoluble extracellular fibrillar deposits. Certain mutations in amyloid prone proteins result in aggressive forms of the disease. β2-microglobulin (β2m), a cell surface protein and transthyretin (TTR), a normal plasma protein, are inherently amyloidogenic. In patients undergoing long-term dialysis, ineffective clearance of β2m from the plasma results in sustained increase of its concentration and its deposition as amyloid. Wild type TTR is the amyloid precursor in senile systemic amyloidosis, a cause of heart failure in the elderly, and various different mutations in the human TTR gene cause the autosomal dominant conditions familial amyloid polyneuropathy and familial amyloid cardiomyopathy. The D76N β2m variant causes highly penetrant hereditary systemic amyloidosis. Similarly, the S52P TTR variant also causes aggressive amyloidosis which is characterised by prominent cardiac ATTR deposits. Animal models for Aβ2m amyloidosis and ATTR amyloidosis have long been sought to enable a better understanding of disease mechanisms and for validation of diagnostic methods and treatments, but previous attempts to model these diseases in vivo have met with limited or no success. The aims of this project were to generate mouse models of: (1) Aβ2m amyloidosis and (2) cardiac ATTR amyloidosis by transgenic expression of these highly amyloidogenic variants. In the work presented here, hβ2mD76N transgenic mice and hTTRS52P transgenic mice were generated. Despite expressing high plasma concentrations of the amyloidogenic proteins, the mice did not spontaneously develop amyloidosis. After priming amyloid deposition with pre formed amyloid fibrils, the hβ2mD76N transgenic mice failed to develop amyloid deposits. It is notable that most of the β2m circulates bound in a complex, potentially limiting the availability of free β2m monomers for conversion into fibrils. In the hTTRS52P transgenic mice, priming of amyloid deposition with amyloid fibrils led to consistent and reproducible development of cardiac ATTR amyloidosis

The effects of short-term JNK inhibition on the survival and growth of aged sympathetic neurons

During the course of normal aging, certain populations of nerve growth factor (NGF)-responsive neurons become selectively vulnerable to cell death. Studies using dissociated neurons isolated from neonates have shown that c-Jun N-terminal kinases (JNKs) are important in regulating the survival and neurite outgrowth of NGF-responsive sympathetic neurons. Unlike neonatal neurons, adult sympathetic neurons are not dependent on NGF for their survival. Moreover, the NGF precursor, proNGF, is neurotoxic for aging but not young adult NGF-responsive neurons. Because of these age-related differences, the effects of JNK inhibition on the survival and growth of sympathetic neurons isolated from aged mice were studied. Aged neurons, as well as glia, were found to be dependent on JNK for their growth but not their survival. Conversely, proNGF neurotoxicity was JNK-dependent and mediated by the p75-interacting protein NRAGE, whereas neurite outgrowth was independent of NRAGE. These results have implications for the potential use of JNK inhibitors as therapies for ameliorating age-related neurodegenerative disease

Plasmin activity promotes amyloid deposition in a transgenic model of human transthyretin amyloidosis

Cardiac ATTR amyloidosis, a serious but much under-diagnosed form of cardiomyopathy, is caused by deposition of amyloid fibrils derived from the plasma protein transthyretin (TTR), but its pathogenesis is poorly understood and informative in vivo models have proved elusive. Here we report the generation of a mouse model of cardiac ATTR amyloidosis with transgenic expression of human TTRS52P. The model is characterised by substantial ATTR amyloid deposits in the heart and tongue. The amyloid fibrils contain both full-length human TTR protomers and the residue 49-127 cleavage fragment which are present in ATTR amyloidosis patients. Urokinase-type plasminogen activator (uPA) and plasmin are abundant within the cardiac and lingual amyloid deposits, which contain marked serine protease activity; knockout of α2-antiplasmin, the physiological inhibitor of plasmin, enhances amyloid formation. Together, these findings indicate that cardiac ATTR amyloid deposition involves local uPA-mediated generation of plasmin and cleavage of TTR, consistent with the previously described mechano-enzymatic hypothesis for cardiac ATTR amyloid formation. This experimental model of ATTR cardiomyopathy has potential to allow further investigations of the factors that influence human ATTR amyloid deposition and the development of new treatments

Comprehensive Analysis of 5-Aminolevulinic Acid Dehydrogenase (ALAD) Variants and Renal Cell Carcinoma Risk among Individuals Exposed to Lead

BACKGROUND: Epidemiologic studies are reporting associations between lead exposure and human cancers. A polymorphism in the 5-aminolevulinic acid dehydratase (ALAD) gene affects lead toxicokinetics and may modify the adverse effects of lead. METHODS: The objective of this study was to evaluate single-nucleotide polymorphisms (SNPs) tagging the ALAD region among renal cancer cases and controls to determine whether genetic variation alters the relationship between lead and renal cancer. Occupational exposure to lead and risk of cancer was examined in a case-control study of renal cell carcinoma (RCC). Comprehensive analysis of variation across the ALAD gene was assessed using a tagging SNP approach among 987 cases and 1298 controls. Occupational lead exposure was estimated using questionnaire-based exposure assessment and expert review. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using logistic regression. RESULTS: The adjusted risk associated with the ALAD variant rs8177796(CT/TT) was increased (OR = 1.35, 95%CI = 1.05-1.73, p-value = 0.02) when compared to the major allele, regardless of lead exposure. Joint effects of lead and ALAD rs2761016 suggest an increased RCC risk for the homozygous wild-type and heterozygous alleles ((GG)OR = 2.68, 95%CI = 1.17-6.12, p = 0.01; (GA)OR = 1.79, 95%CI = 1.06-3.04 with an interaction approaching significance (p(int) = 0.06). No significant modification in RCC risk was observed for the functional variant rs1800435(K68N). Haplotype analysis identified a region associated with risk supporting tagging SNP results. CONCLUSION: A common genetic variation in ALAD may alter the risk of RCC overall, and among individuals occupationally exposed to lead. Further work in larger exposed populations is warranted to determine if ALAD modifies RCC risk associated with lead exposure

Plasmin activity promotes amyloid deposition in a transgenic model of human transthyretin amyloidosis

Cardiac ATTR amyloidosis, a serious but much under-diagnosed form of cardiomyopathy, is caused by deposition of amyloid fibrils derived from the plasma protein transthyretin (TTR), but its pathogenesis is poorly understood and informative in vivo models have proved elusive. Here we report the generation of a mouse model of cardiac ATTR amyloidosis with transgenic expression of human TTRS52P. The model is characterised by substantial ATTR amyloid deposits in the heart and tongue. The amyloid fibrils contain both full-length human TTR protomers and the residue 49-127 cleavage fragment which are present in ATTR amyloidosis patients. Urokinase-type plasminogen activator (uPA) and plasmin are abundant within the cardiac and lingual amyloid deposits, which contain marked serine protease activity; knockout of α2-antiplasmin, the physiological inhibitor of plasmin, enhances amyloid formation. Together, these findings indicate that cardiac ATTR amyloid deposition involves local uPA-mediated generation of plasmin and cleavage of TTR, consistent with the previously described mechano-enzymatic hypothesis for cardiac ATTR amyloid formation. This experimental model of ATTR cardiomyopathy has potential to allow further investigations of the factors that influence human ATTR amyloid deposition and the development of new treatments

Population attributable risk of tobacco and alcohol for upper aerodigestive tract cancer

Tobacco and alcohol are major risk factors for upper aerodigestive tract (UADT) cancer and significant variation is observed in UADT cancer rates across Europe. We have estimated the proportion of UADT cancer burden explained by tobacco and alcohol and how this varies with the incidence rates across Europe, cancer sub-site, gender and age. This should help estimate the minimum residual burden of other risk factors to UADT cancer, including human papillomavirus. We analysed 1981 UADT cancer cases and 1993 controls from the ARCAGE multi-centre study. We estimated the population attributable risk (PAR) of tobacco alone, alcohol alone and their joint effect. Tobacco and alcohol together explained 73% of UADT cancer burden of which nearly 29% was explained by smoking alone, less than 1% due to alcohol on its own and 44% by the joint effect of tobacco and alcohol. Tobacco and alcohol together explained a larger proportion of hypopharyngeal/laryngeal cancer (PAR = 85%) than oropharyngeal (PAR = 74%), esophageal (PAR = 67%) and oral cancer (PAR = 61%). Tobacco and alcohol together explain only about half of the total UADT cancer burden among women. Geographically, tobacco and alcohol explained a larger proportion of UADT cancer in central (PAR = 84%) than southern (PAR = 72%) and western Europe (PAR = 67%). While the majority of the UADT cancers in Europe are due to tobacco or the joint effect of tobacco and alcohol, our results support a significant role for other risk factors in particular, for oral and oropharyngeal cancers and also for UADT cancers in southern and western Europe