From Somatic Variants Toward Precision Oncology: An Investigation of Reporting Practice for Next-Generation Sequencing-Based Circulating Tumor DNA Analysis.

BACKGROUND: With the accelerated development of next-generation sequencing (NGS), identified variants, and targeted therapies, clinicians who confront the complicated and multifarious genetic information may not effectively incorporate NGS-based circulating tumor DNA (ctDNA) analysis into routine patient care. Consequently, standardized ctDNA testing reports are of vital importance. In an effort to guarantee high-quality reporting performance, we conducted an investigation of the current detection and reporting practices for NGS-based ctDNA analysis. MATERIALS AND METHODS: A set of simulated ctDNA samples with known variants at known allelic frequencies and a corresponding case scenario were distributed to 66 genetic testing laboratories for ctDNA analysis. Written reports were collected to evaluate the detection accuracy, reporting integrity, and information sufficiency using 21 predefined criteria. RESULTS: Current reporting practices for NGS-based ctDNA analysis were found to be far from satisfactory, especially regarding testing interpretation and methodological details. Only 42.4% of laboratories reported the results in complete concordance with the expected results. Moreover, 74.2% of reports only listed aberrations with direct and well-known treatment consequences for the tumor type in question. Genetic aberrations for which experimental agents and/or drug access programs are available may thus be overlooked. Furthermore, methodological details for the interpretation of results were missing from the majority of reports (87.9%). CONCLUSION: This proof-of-principle study suggests that the capacity for accurate identification of variants, rational interpretation of genotypes, comprehensive recommendation of potential medications, and detailed description of methodologies need to be further improved before ctDNA analysis can be formally implemented in the clinic. IMPLICATIONS FOR PRACTICE: Accurate, comprehensive, and standardized clinical sequencing reports can help to translate complex genetic information into patient-centered clinical decisions, thereby shepherding precision oncology into daily practice. However, standards, guidelines, and quality requirements for clinical reports of next-generation sequencing (NGS)-based circulating tumor DNA (ctDNA) analysis are currently absent. By using a set of simulated clinical ctDNA samples and a corresponding case scenario, current practices were evaluated to identify deficiencies in clinical sequencing reports of ctDNA analysis. The recommendations provided here may serve as a roadmap for the improved implementation of NGS-based ctDNA analysis in the clinic.

An external quality assurance trial to assess mass spectrometry protein testing facilities for identifying multiple human peptides.

The application of proteomic liquid chromatography mass spectrometry (LC-MS) for identifying proteins and peptides associated with human disease is rapidly growing in clinical diagnostics. However, the ability to accurately and consistently detect disease-associated peptides remains clinically uncertain. Variability in diagnostic testing occurs in part due to the absence of appropriate reference testing materials and standardised clinical guidelines for proteomic testing. In addition, multiple proteomic testing pipelines have not been fully assessed through external quality assurance (EQA). This trial was therefore devised to evaluate the performance of a small number of mass spectrometry (MS) testing facilities to (i) evaluate the EQA material for potential usage in a proteomic quality assurance program, and to (ii) identify key problem areas associated with human peptide testing. Five laboratories were sent six peptide reference testing samples formulated to contain a total of 35 peptides in differing ratios of light (natural) to heavy (labelled) peptides. Proficiency assessment of laboratory data used a modified approach to similarity and dissimilarity testing that was based on Bray-Curtis and Sorensen indices. Proficiency EQA concordant consensus values could not be derived from the assessed data since none of the laboratories correctly identified all reference testing peptides in all samples. However, the produced data may be reflective of specific inter-laboratory differences for detecting multiple peptides since no two testing pipelines used were the same for any laboratory. In addition, laboratory feedback indicated that peptide filtering of the reference material was a common key problem area prior to analysis. These data highlight the importance of an EQA programme for identifying underlying testing issues so that improvements can be made and confidence for clinical diagnostic analysis can be attained.

The emergence of the mitochondrial genome as a partial regulator of nuclear function is providing new insights into the genetic mechanisms underlying age-related complex disease.

Mitochondrial malfunction appears to be intimately associated with age and age-related complex disorders but the precise pathological relevance of such malfunction remains unclear. Mitochondrial, and more specifically bioenergetic, malfunction is commonly encountered in cancer, degenerative disorders and aging. The identification of a mitochondrial-nuclear retrograde signaling pathway in yeast has facilitated the study of the corresponding retrograde signaling mechanisms induced in response to mitochondrial malfunction in mammals including human. Mitochondrial-nuclear crosstalk is critical for the maintenance of cellular homeostasis, and some mitochondrial DNA mutations may perturb crosstalk signaling. However, ascertaining whether mitochondrial malfunction is a cause or a consequence of disease development will be key to determining whether or not impaired crosstalk signaling is of direct pathological and hence therapeutic relevance. Here, we review what is known about the nuclear adaptive compensatory mechanisms induced in response to mitochondrial malfunction. We discuss the role of mitochondrial DNA variants in modulating the penetrance of human inherited disease caused by mutations in the nuclear genome and explore the underlying mechanisms by which they influence the retrograde response. We conclude that mitochondrial DNA variants have the potential to induce molecular signals through the mitochondrial-nuclear crosstalk mechanism, thereby promoting nuclear compensation in response to mitochondrial malfunction. The implications for the development of genetic or pharmaceutical interventions for the treatment of mitochondrial malfunction in complex disease are also explored.

Quaternary protein modeling to predict the function of DNA variation found in human mitochondrial cytochrome c oxidase.

Cytochrome c oxidase (COX) of the electron transport system is thought to be the rate-limiting step in cellular respiration and is found mutated in numerous human pathologies. Here, we employ quaternary three-dimensional (3-D) modeling to construct a model for human COX. The model was used to predict the functional consequences of amino-acid mutations based on phylogenetic conservation of amino acids together with volume and/or steric perturbations, participation in subunit-subunit interfaces and non-covalent energy loss or incompatibilities. These metrics were combined and interpreted for potential functional impact. A notable strength of the 3-D model is that it can interpret and predict the structural consequences of amino-acid variation in all 13 protein subunits. Importantly, the influence of compensatory changes can also be modeled. We examine mutations listed in the human mutation database Mitomap, and in 100 older men, and compare the results from the 3-D model against the automated MutPred web application tool. In combination, these comparisons suggest that the 3-D model predicts more functionally significant mutations than does MutPred. We conclude that the model has useful functional prediction capability but may need modification as functional data on specific mutations becomes known.

Characterisation of a functional intronic polymorphism in the human growth hormone (GH1) gene.

The +1169A allele of the A/T single nucleotide polymorphism (SNP; rs2665802), located within intron 4 of the human growth hormone 1 ( GH1 ) gene, has been associated with reduced levels of circulating GH and insulin-like growth factor 1, a reduced risk of colorectal cancer and a predisposition to osteoporosis. Whether this intronic SNP is itself the functional polymorphism responsible for exerting a direct effect on GH1 gene expression, however, or whether it is instead in linkage disequilibrium with the functional SNP, has been an open question. The evolutionary conservation of the +1169T allele (and the surrounding intronic sequence) in the bovine genome, as well as in primate genomes, is, however, suggestive of its functionality. Although a potential alternative splice site spans the location of the +1169 SNP, polymerase chain reaction-based assays failed to yield any evidence for alternative splicing associated with either allele. To determine whether the +1169 SNP, in different allelic combinations with SNPs at -278 (G/T), -57 (T/G) and +2103 (C/T), exerts a direct effect on gene expression and/or GH secretion, we performed a series of transfections of various GH1 haplotype-expressing constructs into rat GC (somatotroph) cells. The results obtained provided evidence to support the contention that the +1169A allele contributes directly to the observed reduction in both GH1 gene expression and GH secretion. Part of the apparent influence of the +1169A-bearing allele on GH1 gene expression and GH secretion may still, however, be attributable to alleles of additional SNPs in cis to +1169A and located within either the promoter or the 3'-flanking region.

Tumor mutation burden testing: a survey of the International Quality Network for Pathology (IQN Path).

While tumour mutation burden (TMB) is emerging as a possible biomarker for immune-checkpoint inhibitors (ICI), methods for testing have not been standardised as yet. In April 2019, the International Quality Network for Pathology (IQN Path) launched a survey to assess the current practice of TMB testing. Of the 127 laboratories that replied, 69 (54.3%) had already introduced TMB analysis for research purposes and/or clinical applications. Fifty laboratories (72.5%) used targeted sequencing, although a number of different panels were employed. Most laboratories tested formalin-fixed paraffin-embedded material (94.2%), while 18/69 (26%) tested also cell-free DNA. Fifty-five laboratories used both single nucleotide variants and indels for TMB calculation; 20 centers included only non-synonymous variants. In conclusion, the data from this survey indicate that multiple global laboratories were capable of rapidly introducing routine clinical TMB testing. However, the variability of testing methods raises concerns about the reproducibility of results among centers.

Laboratory quality assessment of candidate gene panel testing for acute myeloid leukaemia: a joint ALLG / RCPAQAP initiative.

Accurate classification of acute myeloid leukaemia (AML) has become increasingly reliant on molecular characterisation of this blood cancer. Throughout Australia and New Zealand massively parallel sequencing (MPS) is being adopted by diagnostic laboratories for the routine evaluation of patients with AML. This technology enables the surveying of many genes simultaneously, with many technical advantages over single gene testing approaches. However, there are many variations in wet and dry lab MPS procedures, which raises the prospect of discordant results between laboratories. This study compared the results obtained from MPS testing of ten diagnostic AML bone marrow aspirate samples sent to eight participating laboratories across Australasia. A reassuringly high concordance of 94% was observed with regard to variant detection and characterisation of pathogenicity. The level of discordance observed, although low, demonstrates the need for ongoing assessment of concordance between diagnostic testing laboratories through quality assurance programs.

External Quality Assurance of Current Technology for the Testing of Cancer-Associated Circulating Free DNA Variants.

Liquid biopsy testing is rapidly emerging as a diagnostic means of identifying circulating free DNA (cfDNA) disease-associated variants. However, the reporting of cfDNA variants remains inconsistent due in part to the application of multiple testing pipelines which raise uncertainty about current cfDNA detection efficiency. External quality assurance (EQA) programs are required to monitor, evaluate and help improve laboratory performance for cfDNA variant detection and in clinical interpretation. This study therefore evaluated the performance of diagnostic laboratories currently performing cfDNA testing in China, Australia and New Zealand. A total of 89 laboratories participated in this EQA program. Reference testing material comprised of cfDNA manufactured to contain six different genotypes in four different genes (EGFR, KRAS, BRAF, NRAS). The predicted genotypic variant allelic frequencies ranged between 0.5% - 2.5%. Proficiency testing used a z-score on the laboratory consensus allelic frequency data to compare laboratory performance for the detection of the different genotypes. Allelic frequency genotyping data were received from 88 of the 89 laboratories. Next generation sequencing and digital PCR testing platforms were primarily used by participants in this pilot EQA. The average consensus data for each cfDNA genotype identified allelic frequencies ranging between 0.39% - 4.4%. Z-score proficiency testing found that >92% of clinical laboratories were concordant for detecting the cfDNA variants. The data from this pilot study suggest that current cfDNA testing platforms can detect cfDNA allelic frequency variants from 0.39% and above with high levels of confidence. In addition, these data highlight the importance of laboratories enrolling on EQA programs so that proficiency in cfDNA diagnostic testing can be determined and potential sources of error identified and addressed.

A gene conversion hotspot in the human growth hormone (GH1) gene promoter.

To assess the evolutionary importance of nonallelic (or interlocus) gene conversion for the highly polymorphic human growth hormone (GH1) gene promoter, sequence variation in this region was studied in four different ethnic groups. For 14 SNPs in the proximal GH1 promoter (535 bp), 60 different haplotypes were observed in 577 individuals (156 Britons, 116 Spaniards, 163 West-Africans, 142 Asians). Using a novel coalescence-based statistical test, significant evidence was found in the British, Spanish, and African groups for GH1 having acted as an acceptor of gene conversion, with at least one of the four paralogous GH gene promoters serving as the donor (and specifically GH2 in the Britons and Spaniards). The average gene conversion tract length was estimated to be 84 bp. A gene conversion hotspot was identified, spanning the GH1 transcriptional initiation site (positions -6 to +25). Although these findings serve to highlight the importance of gene conversion for the recent evolution of the human GH1 promoter, its relative frequency does not appear to be related simply to the presence of specific DNA sequence motifs or secondary structures, the degree of homology between GH paralogs, the distance between them, or their transcriptional orientation. The GH1 promoter was also found to be highly polymorphic in chimpanzee but not in macaque. This may reflect the lower degree of pair-wise similarity between the GH1 promoter and its paralogs in macaque (mean, 92.0%) as compared to chimpanzee (93.5%) and human (94.0%), and hence provides further support for the idea of a threshold (perhaps around 92%) below which gene conversion is reduced or abolished.

Application of serial analysis of gene expression to the study of human genetic disease.

Sequence tag analysis using serial analysis of gene expression (SAGE) is a powerful strategy for the quantitative analysis of gene expression in human genetic disorders. SAGE facilitates the measurement of mRNA transcripts and generates a non-biased gene expression profile of normal and pathological disease tissue. In addition, the SAGE technique has the capacity of detecting the expression of novel transcripts allowing for the identification of previously uncharacterised genes, thus providing a unique advantage over the traditional microarray-based approach for expression profiling. The technique has been successful in providing pathological gene expression profiles in a number of common genetic disorders including diabetes, cardiovascular disease, Parkinson disease and Down syndrome. When combined with next generation sequencing platforms, SAGE has the potential to become a more powerful and sensitive technique making it more amenable for diagnostic use. This review will therefore discuss the application of SAGE to several common genetic disorders and will further evaluate its potential use in diagnosing human genetic disease.

Growth hormone (GH1) gene variation and the growth hormone receptor (GHR) exon 3 deletion polymorphism in a West-African population.

Among Europeans, functionally significant GH1 gene variants occur not only in individuals with idiopathic growth hormone (GH) deficiency and/or short stature but also fairly frequently in the general population. To assess the generality of these findings, 163 individuals from Benin, West Africa were screened for mutations and polymorphisms in their GH1 genes. A total of 37 different sequence variants were identified in the GH1 gene region, 24 of which occurred with a frequency of >1%. Although four of these variants were novel missense substitutions (Ala13Val, Arg19His, Phe25Tyr and Ser95Arg), none of these had any measurable effect on either GH function or secretion in vitro. Some 37 different GH1 promoter haplotypes were identified, 23 of which are as yet unreported in Europeans. The mean in vitro expression level of the GH1 promoter haplotypes observed in the African population was significantly higher than that previously measured in Britons (p<0.001). A gene conversion in the GH1 promoter, previously reported in a single individual of British origin, was found to occur at polymorphic frequency (5%) in the West-African population and was associated with a 1.7-fold increase in promoter activity relative to the wild-type. The d3 allele of the GHR exon 3 deletion polymorphism, known to be associated with increased GH responsiveness, was also found to occur at an elevated frequency in these individuals from Benin. We speculate that both elevated GH1 gene expression and increased GHR-mediated GH responsiveness may constitute adaptive responses to the effects of scarce food supply in this West-African population since increased circulating GH appears to form part of a physiological response to nutritional deprivation.

High rates of variation in HLA-DQ2/DQ8 testing for coeliac disease: results from an RCPAQAP pilot program.

AIM: Coeliac disease(CD) is a highly prevalent, gluten-dependent, autoimmune enteropathy. While the diagnosis is based on serological and histological criteria, genotyping of the human leucocyte antigens (HLA) DQ2 and DQ8 has been shown to have substantial clinical utility, especially in excluding the diagnosis in patients who do not carry either antigen. As a result, HLA genotyping is now being performed by more laboratories and has recently become one of the most frequently requested genetic tests in Australia. To date, there has been little scrutiny on the accuracy and reporting of results by laboratories new to HLA typing. In response to clinician feedback that identified potentially clinically significant discrepancies in HLA typing results, the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) undertook a pilot study to assess laboratory performance in the detection of HLA-DQ2/DQ8 and their associated HLA-DQA1 and HLA-DQB1 alleles. METHODS: DNA was extracted from 5 patients and sent to 10 laboratories for external quality assurance (EQA) testing. Laboratories were assessed for reporting in genotyping, interpretation and methodology. RESULTS: Our findings showed that at least 80% of laboratories underperform with respect to recommended guidelines for HLA typing and reporting for CD, with 40% of laboratories failing to provide any clinical interpretation or full genotyping data. This suboptimal level of reporting may lead to ambiguities for downstream clinical interpretation that may compromise patient management. CONCLUSIONS: These findings highlight the importance of adherence to standardised guidelines for optimal performance and reporting of HLA results and substantiate the need for EQA and proficiency testing for laboratories providing this service.

The Influence of Macronutrients on Splanchnic and Hepatic Lymphocytes in Aging Mice.

There is a strong association between aging, diet, and immunity. The effects of macronutrients and energy intake on splanchnic and hepatic lymphocytes were studied in 15 month old mice. The mice were ad-libitum fed 1 of 25 diets varying in the ratios and amounts of protein, carbohydrate, and fat over their lifetime. Lymphocytes in liver, spleen, Peyers patches, mesenteric lymph nodes, and inguinal lymph nodes were evaluated using flow cytometry. Low protein intake reversed aging changes in splenic CD4 and CD8 T cells, CD4:CD8 T cell ratio, memory/effector CD4 T cells and naïve CD4 T cells. A similar influence of total caloric intake in these ad-libitum fed mice was not apparent. Protein intake also influenced hepatic NK cells and B cells, while protein to carbohydrate ratio influenced hepatic NKT cells. Hepatosteatosis was associated with increased energy and fat intake and changes in hepatic Tregs, effector/memory T, and NK cells. Hepatic NK cells were also associated with body fat, glucose tolerance, and leptin levels while hepatic Tregs were associated with hydrogen peroxide production by hepatic mitochondria. Dietary macronutrients, particularly protein, influence splanchnic lymphocytes in old age, with downstream associations with mitochondrial function, liver pathology, and obesity-related phenotype.

Human growth hormone 1 (GH1) gene expression: complex haplotype-dependent influence of polymorphic variation in the proximal promoter and locus control region.

The proximal promoter region of the human pituitary expressed growth hormone (GH1) gene is highly polymorphic, containing at least 15 single nucleotide polymorphisms (SNPs). This variation is manifest in 40 different haplotypes, the high diversity being explicable in terms of gene conversion, recurrent mutation, and selection. Functional analysis showed that 12 haplotypes were associated with a significantly reduced level of reporter gene expression whereas 10 haplotypes were associated with a significantly increased level. The former tend to be more prevalent in the general population than the latter (p<0.01), possibly as a consequence of selection. Although individual SNPs contributed to promoter strength in a highly interactive and non-additive fashion, haplotype partitioning was successful in identifying six SNPs as major determinants of GH1 gene expression. The prediction and functional testing of hitherto unobserved super-maximal and sub-minimal promoter haplotypes was then used to test the efficacy of the haplotype partitioning approach. Electrophoretic mobility shift assays demonstrated that five SNP sites exhibit allele-specific protein binding. An association was noted between adult height and the mean in vitro expression value corresponding to an individual's GH1 promoter haplotype combination (p=0.028) although only 3.3% of the variance of adult height was found to be explicable by reference to this parameter. Three additional SNPs, identified within sites I and II of the upstream locus control region (LCR), were ascribed to three distinct LCR haplotypes. A series of LCR-GH1 proximal promoter constructs were used to demonstrate that 1) the LCR enhanced proximal promoter activity by up to 2.8-fold depending upon proximal promoter haplotype, and that 2) the activity of a given proximal promoter haplotype was also differentially enhanced by different LCR haplotypes. The genetic basis of inter-individual differences in GH1 gene expression thus appears to be extremely complex.

Characterization of the somatic mutational spectrum of the neurofibromatosis type 1 (NF1) gene in neurofibromatosis patients with benign and malignant tumors.

One of the main features of neurofibromatosis type 1 (NF1) is benign neurofibromas, 10-20% of which become transformed into malignant peripheral nerve sheath tumors (MPNSTs). The molecular basis of NF1 tumorigenesis is, however, still unclear. Ninety-one tumors from 31 NF1 patients were screened for gross changes in the NF1 gene using microsatellite/restriction fragment length polymorphism (RFLP) markers; loss of heterozygosity (LOH) was found in 17 out of 91 (19%) tumors (including two out of seven MPNSTs). Denaturing high performance liquid chromatography (DHPLC) was then used to screen 43 LOH-negative and 10 LOH-positive tumors for NF1 microlesions at both RNA and DNA levels. Thirteen germline and 12 somatic mutations were identified, of which three germline (IVS7-2A>G, 3731delT, 6117delG) and eight somatic (1888delG, 4374-4375delCC, R2129S, 2088delG, 2341del18, IVS27b-5C>T, 4083insT, Q519P) were novel. A mosaic mutation (R2429X) was also identified in a neurofibroma by DHPLC analysis and cloning/sequencing. The observed somatic and germline mutational spectra were similar in terms of mutation type, relative frequency of occurrence, and putative underlying mechanisms of mutagenesis. Tumors lacking mutations were screened for NF1 gene promoter hypermethylation but none were found. Microsatellite instability (MSI) analysis revealed MSI in five out of 11 MPNSTs as compared to none out of 70 neurofibromas (p=1.8 x 10(-5)). The screening of seven MPNSTs for subtle mutations in the CDKN2A and TP53 genes proved negative, although the screening of 11 MPNSTs detected LOH involving either the TP53 or the CDKN2A gene in a total of four tumors. These findings are consistent with the view that NF1 tumorigenesis is a complex multistep process involving a variety of different types of genetic defect at multiple loci.

Novel mutations of the growth hormone 1 (GH1) gene disclosed by modulation of the clinical selection criteria for individuals with short stature.

Subtle mutations in the growth hormone 1 (GH1) gene have been regarded as a comparatively rare cause of short stature. Such lesions were sought in a group of 41 individuals selected for short stature, reduced height velocity, and bone age delay; a group of 11 individuals with short stature and idiopathic growth hormone deficiency (IGHD); and a group of 154 controls. Heterozygous mutations were identified in all three groups but disproportionately in the individuals with short stature, both with (odds ratio 25.2; 95% CI, 5.1-132.2) and without (odds ratio 3.6; 95% CI, 1.0-12.9) IGHD. Twenty-four novel GH1 gene lesions were found. Thirteen novel missense mutations were characterized by assaying the signal transduction activity of in vitro expressed variants; six (T27I, K41R, N47D, S71F, S108R, and T175A) exhibited a reduced ability to activate the JAK/STAT pathway. Molecular modeling suggested that both K41R and T175A might compromise GH receptor binding. Seven GH variants (R16C, K41R, S71F, E74K, Q91L, S108C, and a functional polymorphism, V110I) manifested reduced secretion in rat pituitary cells after allowance had been made for the level of expression attributable to the associated GH1 proximal promoter haplotype. A further leader peptide variant (L-11P) was not secreted. Eleven novel mutations in the GH1 gene promoter were assessed by reporter gene assay but only two, including a GH2 gene-templated gene conversion, were found to be associated with a significantly reduced level of expression. Finally, a novel intron 2 acceptor splice-site mutation, detected in a family with autosomal dominant type II IGHD, was shown to lead to the skipping of exon 3 from the GH1 transcript. A total of 15 novel GH1 gene mutations were thus considered to be of probable phenotypic significance. Such lesions are more prevalent than previously recognized and although most may be insufficient on their own to account for the observed clinical phenotype, they are nevertheless likely to play a contributory role in the etiology of short stature.

A novel dysfunctional growth hormone variant (Ile179Met) exhibits a decreased ability to activate the extracellular signal-regulated kinase pathway.

The pituitary-expressed GH1 gene was screened for mutation in a group of 74 children with familial short stature. Two novel mutations were identified: an Ile179Met substitution and a -360A-->G promoter variant. The Ile179Met variant was shown to exhibit a similar degree of resistance to proteolysis as wild-type GH, indicating that the introduction of Met does not cause significant misfolding. Secretion of Ile179Met GH from rat pituitary cells was also similar to that of wild type. Although receptor binding studies failed to show any difference in binding characteristics, molecular modeling studies suggested that the Ile179Met substitution might nevertheless perturb interactions between GH and the GH receptor loop containing the hotspot residue Trp169, thereby affecting signal transduction. The ability of the Ile179Met variant to activate a signal transducer and activator of transcription (STAT) 5-responsive luciferase reporter gene and induce phosphorylation of STAT 5 and ERK was therefore studied. In contrast to its ability to activate STAT 5 normally, activation of ERK by the Ile179Met variant was reduced to half that observed with wild type. Although differential effects on the activation of distinct signaling pathways by a mutant receptor agonist are unprecedented, these findings also suggest that the ERK pathway could play a role in mediating the action of GH.

Alpha-synuclein transmission and mitochondrial toxicity in primary human foetal enteric neurons in vitro.

Parkinson's disease (PD) is a multicentred neurodegenerative disorder characterised by the accumulation and aggregation of alpha-synuclein (α-syn) in several parts of the central nervous system. However, it is well established that PD can generate symptoms of constipation and other gastrointestinal problems and α-syn containing lesions have been identified in intestinal nerve cells. In this study, we show that α-syn can be taken up and accumulate in primary human foetal enteric neurons from the gastrointestinal tract and can be transferred between foetal enteric neurons. Impaired proteosomal/lysosomal degradation can promote the uptake and accumulation of α-syn in enteric neurons. Enteric neurons exposed to α-syn can also lead to impaired mitochondrial complex I activity, reduced mitochondrial function, and NAD(+) depletion culminating in cell death via energy restriction. These findings demonstrate neuron-to-neuron transmission of α-syn in enteric neurons, providing renewed evidence for Braak's hypothesis and the aetiology of PD.

Uptake and mitochondrial dysfunction of alpha-synuclein in human astrocytes, cortical neurons and fibroblasts.

The accumulation and aggregation of alpha-synuclein (α-syn) in several tissue including the brain is a major pathological hallmark in Parkinson's disease (PD). In this study, we show that α-syn can be taken up by primary human cortical neurons, astrocytes and skin-derived fibroblasts in vitro. Our findings that brain and peripheral cells exposed to α-syn can lead to impaired mitochondrial function, leading to cellular degeneration and cell death, provides additional evidence for the involvement of mitochondrial dysfunction as a mechanism of toxicity of α-syn in human cells.

Review: quantifying mitochondrial dysfunction in complex diseases of aging.

There is accumulating evidence that mitochondrial respiratory malfunction is associated with aging-associated complex diseases. However, progress in our understanding of these diseases has been hampered by the sensitivity and throughput of systems employed to quantify dysfunction and inherent limitations of the biological systems studied. In this review, we describe and contrast two methodologies that have been developed for measuring mitochondrial function to address the need for improved sensitivity and increased throughput. We then consider the utility of each methodology in studying three biological systems: isolated mitochondria, cultured cells, and cell fibers and tissues. Finally, we discuss the application of each methodology in the study of mitochondrial dysfunction in Alzheimer's disease, type 2 diabetes mellitus, and aging-associated autophagy impairment and mitochondrial malfunction. We conclude that the methodologies are complementary, and researchers may need to examine multiple biological systems to unravel complex diseases of aging.