Mitochondrial dysfunction in dopamine neurons : implications for Parkinson’s disease

Mitochondria are essential for cellular homeostasis and contain the respiratory chain (RC). Decreased mitochondrial function is associated with ageing, as exemplified by the finding of a mosaic pattern of RC-deficient cells in aged tissues. Parkinson’s disease (PD) is a common age-related disorder characterized by loss of midbrain dopamine (DA) neurons and formation of intracellular inclusions. A number of observations suggest a role for mitochondrial dysfunction in the pathophysiology of PD: (1) toxins linked to PD have been shown to impair RC function, (2) reduced RC enzyme activities are found in patient tissues, (3) the proportion of DA neurons that are RC-deficient, due to accumulation of mtDNA deletions, is higher in PD patients than in controls, and (4) an inherited form of PD is caused by loss-of-function mutations in the gene for Parkin, an E3 ubiquitin ligase reported to facilitate clearance of defective mitochondria. In this thesis, experimental genetics in mice have been used to study consequences of mitochondrial dysfunction in the brain, with particular focus on DA neurons. First, we addressed the role of mosaic RC deficiency in the brain by creating chimeric mice with a mixture of normal and RC-deficient forebrain neurons. A low proportion (>20%) of respiratory chain-deficient forebrain neurons was sufficient to cause symptoms. On the one hand, surrounding normal neurons could prevent mortality and delay the onset of symptoms. On the other hand, RC-deficient neurons could induce death of surrounding normal neurons by a trans-neuronal degeneration mechanism. We also developed so-called ‘MitoPark’ mice, which have DA-specific disruption of Tfam, a gene critical for maintenance and expression of mtDNA. MitoPark mice have severe RC deficiency in DA neurons and develop slow, progressive loss of DA neurons accompanied by motor symptoms resembling those seen in PD. To study changes in mitochondrial morphology and distribution, we developed a novel reporter mouse for cell type-specific labeling of mitochondria. We found that mitochondria in RC-deficient neurons fragmented and concomitantly formed severely enlarged mitochondrial bodies in the soma and proximal dendrites. Mitochondria in distal axon segments had normal morphology, but the RC-deficient neurons had an impaired anterograde supply of new mitochondria to their axon terminals. We did not find support for a role of Parkin in MitoPark DA neurons, as overexpressed Parkin was not recruited to the aberrant mitochondria and the absence of Parkin did not affect their clearance. Finally, we studied the consequences of a complex I defect in DA neurons. Disruption of the complex I subunit gene Ndufs4 resulted in a mild complex I deficiency, which did not cause degeneration of DA neurons or PD-like symptoms. Nevertheless, we found increased levels of DA metabolites and impaired DA release at the level of the axon terminals, compatible with early changes seen in PD. Ndufs4 knockout DA neurons were in addition more vulnerable to toxic insult. In summary, these results support a role for mitochondrial dysfunction in PD and show that RC function is important for axonal mitochondrial transport and synaptic DA release

A transcriptional timetable of autumn senescence

BACKGROUND: We have developed genomic tools to allow the genus Populus (aspens and cottonwoods) to be exploited as a full-featured model for investigating fundamental aspects of tree biology. We have undertaken large-scale expressed sequence tag (EST) sequencing programs and created Populus microarrays with significant gene coverage. One of the important aspects of plant biology that cannot be studied in annual plants is the gene activity involved in the induction of autumn leaf senescence. RESULTS: On the basis of 36,354 Populus ESTs, obtained from seven cDNA libraries, we have created a DNA microarray consisting of 13,490 clones, spotted in duplicate. Of these clones, 12,376 (92%) were confirmed by resequencing and all sequences were annotated and functionally classified. Here we have used the microarray to study transcript abundance in leaves of a free-growing aspen tree (Populus tremula) in northern Sweden during natural autumn senescence. Of the 13,490 spotted clones, 3,792 represented genes with significant expression in all leaf samples from the seven studied dates. CONCLUSIONS: We observed a major shift in gene expression, coinciding with massive chlorophyll degradation, that reflected a shift from photosynthetic competence to energy generation by mitochondrial respiration, oxidation of fatty acids and nutrient mobilization. Autumn senescence had much in common with senescence in annual plants; for example many proteases were induced. We also found evidence for increased transcriptional activity before the appearance of visible signs of senescence, presumably preparing the leaf for degradation of its components

Calsyntenin-3 Interacts With Both α- And β-Neurexins in the Regulation of Excitatory Synaptic Innervation in Specific Schaffer Collateral Pathways

Calsyntenin-3 (Clstn3) is a postsynaptic adhesion molecule that induces presynaptic differentiation via presynaptic neurexins (Nrxns), but whether Nrxns directly bind to Clstn3 has been a matter of debate. Here, using LC-MS/MS-based protein analysis, confocal microscopy, RNAscope assays, and electrophysiological recordings, we show that β-Nrxns directly interact via their LNS domain with Clstn3 and Clstn3 cadherin domains. Expression of splice site 4 (SS4) insert-positive β-Nrxn variants, but not insert-negative variants, reversed the impaired Clstn3 synaptogenic activity observed in Nrxn-deficient neurons. Consistently, Clstn3 selectively formed complexes with SS4-positive Nrxns in vivo Neuron-specific Clstn3 deletion caused significant reductions in number of excitatory synaptic inputs. Moreover, expression of Clstn3 cadherin domains in CA1 neurons of Clstn3 conditional knockout mice rescued structural deficits in excitatory synapses, especially within the stratum radiatum layer. Collectively, our results suggest that Clstn3 links to SS4-positive Nrxns to induce presynaptic differentiation and orchestrate excitatory synapse development in specific hippocampal neural circuits, including Schaffer collateral afferents. © 2020 Kim et al.1

Microarray data quality analysis: lessons from the Arabidopsis functional genomics consortium

Genome-wide expression profiling with DNA microarrays has and will provide a great deal of data to the plant scientific community. However, reliability concerns have required the development data quality tests for common systematic biases. Fortunately, most large-scale systematic biases are detectable and some are correctable by normalization. Technical replication experiments and statistical surveys indicate that these biases vary widely in severity and appearance. As a result, no single normalization or correction method currently available is able to address all the issues. However, careful sequence selection, array design, experimental design and experimental annotation can substantially improve the quality and biological of microarray data. In this review, we discuss these issues with reference to examples from the Arabidopsis Functional Genomics Consortium (AFGC) microarray project

EST data suggest that poplar is an ancient polyploid

We analysed the publicly available expressed sequence tag (EST) collections for the genus Populus to examine whether evidence can be found for large-scale gene-duplication events in the evolutionary past of this genus. The ESTs were clustered into unigenes for each poplar species examined. Gene families were constructed for all proteins deduced from these unigenes, and K-S dating was performed on all paralogs within a gene family. The fraction of paralogs was then plotted against the K-S values, which resulted in a distribution reflecting the age of duplicated genes in poplar. Sufficient EST data were available for seven different poplar species spanning four of the six sections of the genus Populus. For all these species, there was evidence that a large-scale gene-duplication event had occurred. From our analysis it is clear that all poplar species have shared the same large-scale gene-duplication event, suggesting that this event must have occurred in the ancestor of poplar, or at least very early in the evolution of the Populus genus

Iterative linear regression by sector: renormalization of cDNA microarray data and cluster analysis weighted by cross homology.

Two-color DNA microarray data has proven valuable in high-throughput expression profiling. However microarray expression ratios (log 2 ratios) are subject to measurement error from multiple causes. Transcript abundance is expected to be a linear function of signal intensity (y = x) where the typical gene is nonresponsive. Once linearity is confirmed, applying the model by fitting log-scale data with simple linear regression reduces the standard deviation of the log 2 ratios. After which fewer genes are selected by filtering methods. Comparing the residuals of regression to leverage measures can identify the best candidate genes. Spatial bias in log 2 ratio, defined by printing pin and detected by ANOVA, can be another source of measurement error. Independently applying the linear normalization method to the data from each pin can easily eliminate this error. Less easily addressed is the problem of cross-homology which is expected to correlate to cross-hybridization. Pair-wise comparison of genes demonstrate that genes with similar sequences are measured as having similar expression. While this bias cannot be easily eliminated, the effect this probable cross-hybridization can be minimized in clustering by weighting methods introduced here

Gene Expression in Autumn Leaves

Two cDNA libraries were prepared, one from leaves of a field-grown aspen (Populus tremula) tree, harvested just before any visible sign of leaf senescence in the autumn, and one from young but fully expanded leaves of greenhouse-grown aspen (Populus tremula × tremuloides). Expressed sequence tags (ESTs; 5,128 and 4,841, respectively) were obtained from the two libraries. A semiautomatic method of annotation and functional classification of the ESTs, according to a modified Munich Institute of Protein Sequences classification scheme, was developed, utilizing information from three different databases. The patterns of gene expression in the two libraries were strikingly different. In the autumn leaf library, ESTs encoding metallothionein, early light-inducible proteins, and cysteine proteases were most abundant. Clones encoding other proteases and proteins involved in respiration and breakdown of lipids and pigments, as well as stress-related genes, were also well represented. We identified homologs to many known senescence-associated genes, as well as seven different genes encoding cysteine proteases, two encoding aspartic proteases, five encoding metallothioneins, and 35 additional genes that were up-regulated in autumn leaves. We also indirectly estimated the rate of plastid protein synthesis in the autumn leaves to be less that 10% of that in young leaves