Cerebral small vessel disease burden and cognitive and functional outcomes after stroke: A multicenter prospective cohort study.

INTRODUCTION: It remains unknown whether the global small vessel disease (SVD) burden predicts post-stroke outcomes. METHODS: In a prospective multicenter study of 666 ischemic and hemorrhagic stroke patients, we quantified magnetic resonance imaging (MRI)-based SVD markers (lacunes, white matter hyperintensities, microbleeds, perivascular spaces) and explored associations with 6- and 12-month cognitive (battery of 15 neuropsychological tests) and functional (modified Rankin scale) outcomes. RESULTS: A global SVD score (range 0-4) was associated with cognitive impairment; worse performance in executive function, attention, language, and visuospatial ability; and worse functional outcome across a 12-month follow-up. Although the global SVD score did not improve prediction, individual SVD markers, assessed across their severity range, improved the calibration, discrimination, and reclassification of predictive models including demographic, clinical, and other imaging factors. DISCUSSION: SVD presence and severity are associated with worse cognitive and functional outcomes 12 months after stroke. Assessing SVD severity may aid prognostication for stroke patients. HIGHLIGHTS: In a multi-center cohort, we explored associations of small vessel disease (SVD) burden with stroke outcomes. SVD burden associates with post-stroke cognitive and functional outcomes. A currently used score of SVD burden does not improve the prediction of poor outcomes. Assessing the severity of SVD lesions adds predictive value beyond known predictors. To add predictive value in assessing SVD in stroke patients, SVD burden scores should integrate lesion severity.

Functional complementation analyses reveal that the single PRAT family protein of trypanosoma brucei is a divergent homolog of Tim17 in saccharomyces cerevisiae.

Trypanosoma brucei, a parasitic protozoan that causes African trypanosomiasis, possesses a single member of the presequence and amino acid transporter (PRAT) protein family, which is referred to as TbTim17. In contrast, three homologous proteins, ScTim23, ScTim17, and ScTim22, are found in Saccharomyces cerevisiae and higher eukaryotes. Here, we show that TbTim17 cannot rescue Tim17, Tim23, or Tim22 mutants of S. cerevisiae. We expressed S. cerevisiae Tim23, Tim17, and Tim22 in T. brucei. These heterologous proteins were properly imported into mitochondria in the parasite. Further analysis revealed that although ScTim23 and ScTim17 were integrated into the mitochondrial inner membrane and assembled into a protein complex similar in size to TbTim17, only ScTim17 was stably associated with TbTim17. In contrast, ScTim22 existed as a protease-sensitive soluble protein in the T. brucei mitochondrion. In addition, the growth defect caused by TbTim17 knockdown in T. brucei was partially restored by the expression of ScTim17 but not by the expression of either ScTim23 or ScTim22, whereas the expression of TbTim17 fully complemented the growth defect caused by TbTim17 knockdown, as anticipated. Similar to the findings for cell growth, the defect in the import of mitochondrial proteins due to depletion of TbTim17 was in part restored by the expression of ScTim17 but was not complemented by the expression of either ScTim23 or ScTim22. Together, these results suggest that TbTim17 is divergent compared to ScTim23 but that its function is closer to that of ScTim17. In addition, ScTim22 could not be sorted properly in the T. brucei mitochondrion and thus failed to complement the function of TbTim17.

Cooperation of TOM and TIM23 complexes during translocation of proteins into mitochondria.

Translocation of the majority of mitochondrial proteins from the cytosol into mitochondria requires the cooperation of TOM and TIM23 complexes in the outer and inner mitochondrial membranes. The molecular mechanisms underlying this cooperation remain largely unknown. Here, we present biochemical and genetic evidence that at least two contacts from the side of the TIM23 complex play an important role in TOM-TIM23 cooperation in vivo. Tim50, likely through its very C-terminal segment, interacts with Tom22. This interaction is stimulated by translocating proteins and is independent of any other TOM-TIM23 contact known so far. Furthermore, the exposure of Tim23 on the mitochondrial surface depends not only on its interaction with Tim50 but also on the dynamics of the TOM complex. Destabilization of the individual contacts reduces the efficiency of import of proteins into mitochondria and destabilization of both contacts simultaneously is not tolerated by yeast cells. We conclude that an intricate and coordinated network of protein-protein interactions involving primarily Tim50 and also Tim23 is required for efficient translocation of proteins across both mitochondrial membranes.

Role of the import motor in insertion of transmembrane segments by the mitochondrial TIM23 complex.

The TIM23 complex mediates translocation of proteins across, and their lateral insertion into, the mitochondrial inner membrane. Translocation of proteins requires both the membrane-embedded core of the complex and its ATP-dependent import motor. Insertion of some proteins, however, occurs in the absence of ATP, questioning the need for the import motor during lateral insertion. We show here that the import motor associates with laterally inserted proteins even when its ATPase activity is not required. Furthermore, our results suggest a role for the import motor in lateral insertion. Thus, the import motor is involved in ATP-dependent translocation and ATP-independent lateral insertion.

The conformational dynamics of the mitochondrial Hsp70 chaperone.

Heat shock proteins 70 (Hsp70) represent a ubiquitous and conserved family of molecular chaperones involved in a plethora of cellular processes. The dynamics of their ATP hydrolysis-driven and cochaperone-regulated conformational cycle are poorly understood. We used fluorescence spectroscopy to analyze, in real time and at single-molecule resolution, the effects of nucleotides and cochaperones on the conformation of Ssc1, a mitochondrial member of the family. We report that the conformation of its ADP state is unexpectedly heterogeneous, in contrast to a uniform ATP state. Substrates are actively involved in determining the conformation of Ssc1. The J protein Mdj1 does not interact transiently with the chaperone, as generally believed, but rather is released slowly upon ATP hydrolysis. Analysis of the major bacterial Hsp70 revealed important differences between highly homologous members of the family, possibly explaining tuning of Hsp70 chaperones to meet specific functions in different organisms and cellular compartments.

A protein kinase family in Arabidopsis phosphorylates chloroplast precursor proteins.

A serine/threonine protein kinase that is able to phosphorylate chloroplast-destined precursor proteins was purified from leaf extract of Arabidopsis thaliana and was identified by mass spectrometry. The protein kinase, encoded by AT2G17700, belongs to a small protein family comprising in addition AT4G35780 and AT4G38470. All three proteins were expressed heterologously in Escherichia coli and characterized with regard to their properties in precursor protein phosphorylation. They were able to phosphorylate several chloroplast-destined precursor proteins within their cleavable presequences. In contrast, a mitochondria-destined precursor protein was not a substrate for these kinases. For all three enzymes, the phosphorylation reaction was specific for ATP with apparent K(m) values between 14 and 67 microM. They did not utilize other NTPs nor were those able to compete for ATP in the reaction. An excess of ADP was able to inhibit ATP-dependent phosphorylation. Furthermore, all three kinases exhibited autophosphorylation. The protein kinases described here could represent subunits of a regulatory network involved in the cytosolic events of chloroplast protein import.