Cystatin C, cognition, and brain MRI findings in 90+-year-olds.

Chronic kidney disease is emerging as a novel risk factor for cerebrovascular disease, but this association remains largely unexplored in older adults. Cystatin C is a more accurate measure than creatinine of kidney function in the elderly. We evaluated cystatin C, cognitive function, and brain imaging in 193 participants from The 90+ Study neuroimaging component. The mean age was 93.9 years; 61% were women. Mean cystatin C was 1.62 mg/L with estimated glomerular filtration rate 39.2 mL/min/1.73 m2. Performance on measures of global cognition, executive function, and visual-spatial ability declined at higher tertiles of cystatin C (lower kidney function). Higher cystatin C was significantly associated with infratentorial microbleeds and lower gray matter volume. Adjusted risk of incident dementia was increased in the middle and high cystatin C tertile groups compared with the low group (hazard ratio in highest tertile 3.81 [95% confidence interval 1.14-12.7]), which appeared to be explained in part by the presence of cerebral microbleeds. Overall, cystatin C was associated with cognitive performance, brain imaging pathology, and decline to dementia in this oldest-old cohort.

Neuropsychological Test Norms in Cognitively Intact Oldest-Old.

OBJECTIVES: Individuals aged 90 or older (oldest-old), the fastest growing segment of the population, are at increased risk of developing cognitive impairment compared with younger old. Neuropsychological evaluation of the oldest-old is important yet challenging in part because of the scarcity of test norms for this group. We provide neuropsychological test norms for cognitively intact oldest-old. METHODS: Test norms were derived from 403 cognitively intact participants of The 90+ Study, an ongoing study of aging and dementia in the oldest-old. Cognitive status of intact oldest-old was determined at baseline using cross-sectional approach. Individuals with cognitive impairment no dementia or dementia (according to DSM-IV criteria) were excluded. Participants ranged in age from 90 to 102 years (mean=94). The neuropsychological battery included 11 tests (Mini-Mental Status Examination, Modified Mini-Mental State Examination, Boston Naming Test - Short Form, Letter Fluency Test, Animal Fluency Test, California Verbal Learning Test-II Short Form, Trail Making Tests A/B/C, Digit Span Forward and Backwards Test, Clock Drawing Test, CERAD Construction Subtests), and the Geriatric Depression Scale. RESULTS: Data show significantly lower scores with increasing age on most tests. Education level, sex, and symptoms of depression were associated with performance on several tests after accounting for age. CONCLUSIONS: Provided test norms will help to distinguish cognitively intact oldest-old from those with cognitive impairment. (JINS, 2019, 25, 530-545).

DNA-binding specificity and in vivo targets of Caenorhabditis elegans nuclear factor I.

The conserved nuclear factor I (NFI) family of transcription factors is unique to animals and essential for mammalian development. The Caenorhabditis elegans genome encodes a single NFI family member, whereas vertebrate genomes encode 4 distinct NFI protein subtypes (A, B, C, and X). NFI-1-deficient worms exhibit abnormalities, including reduced lifespan, defects in movement and pharyngeal pumping, and delayed egg-laying. To explore the functional basis of these phenotypes, we sought to comprehensively identify NFI-1-bound loci in C. elegans. We first established NFI-1 DNA-binding specificity using an in vitro DNA-selection strategy. Analysis yielded a consensus motif of TTGGCA(N)(3)TGCCAA, which occurs 586 times in the genome, a 100-fold higher frequency than expected. We next asked which sites were occupied by NFI-1 in vivo by performing chromatin immunoprecipitation of NFI-1 followed by microarray hybridization. Only 55 genomic locations were identified, an unexpectedly small target set. In vivo NFI-1 binding sites tend to be upstream of genes involved in core cellular processes, such as chromatin remodeling, mRNA splicing, and translation. Remarkably, 59 out of 70 (84%) of the C. briggsae orthologs of the identified targets contain conserved NFI binding sites in their promoters. These experiments provide a foundation for understanding how NFI-1 is recruited to unexpectedly few in vivo sites to perform its developmental functions, despite a vast over-representation of its binding motif.

The genomic distribution and function of histone variant HTZ-1 during C. elegans embryogenesis.

In all eukaryotes, histone variants are incorporated into a subset of nucleosomes to create functionally specialized regions of chromatin. One such variant, H2A.Z, replaces histone H2A and is required for development and viability in all animals tested to date. However, the function of H2A.Z in development remains unclear. Here, we use ChIP-chip, genetic mutation, RNAi, and immunofluorescence microscopy to interrogate the function of H2A.Z (HTZ-1) during embryogenesis in Caenorhabditis elegans, a key model of metazoan development. We find that HTZ-1 is expressed in every cell of the developing embryo and is essential for normal development. The sites of HTZ-1 incorporation during embryogenesis reveal a genome wrought by developmental processes. HTZ-1 is incorporated upstream of 23% of C. elegans genes. While these genes tend to be required for development and occupied by RNA polymerase II, HTZ-1 incorporation does not specify a stereotypic transcription program. The data also provide evidence for unexpectedly widespread independent regulation of genes within operons during development; in 37% of operons, HTZ-1 is incorporated upstream of internally encoded genes. Fewer sites of HTZ-1 incorporation occur on the X chromosome relative to autosomes, which our data suggest is due to a paucity of developmentally important genes on X, rather than a direct function for HTZ-1 in dosage compensation. Our experiments indicate that HTZ-1 functions in establishing or maintaining an essential chromatin state at promoters regulated dynamically during C. elegans embryogenesis.

Neuropsychological data in nondemented oldest old: the 90+ Study.

Although the oldest old are the fastest growing segment of the population, little is known about their cognitive performance. Our aim was to compile a relatively brief test battery that could be completed by a majority of individuals aged 90 or over, compensates for sensory losses, and incorporates previously validated, standardized, and accessible instruments. Means, standard deviations, and percentiles for 10 neuropsychological tests covering multiple cognitive domains are reported for 339 nondemented members of the 90+ Study. Cognitive performance declined with age for two-thirds of the tests. Performance on some tests was also affected by gender, education, and depression scores.

X chromosome repression by localization of the C. elegans dosage compensation machinery to sites of transcription initiation.

Among organisms with chromosome-based mechanisms of sex determination, failure to equalize expression of X-linked genes between the sexes is typically lethal. In C. elegans, XX hermaphrodites halve transcription from each X chromosome to match the output of XO males. Here, we mapped the binding location of the condensin homolog DPY-27 and the zinc finger protein SDC-3, two components of the C. elegans dosage compensation complex (DCC). We observed strong foci of DCC binding on X, surrounded by broader regions of localization. Binding foci, but not adjacent regions of localization, were distinguished by clusters of a 10-bp DNA motif, suggesting a recruitment-and-spreading mechanism for X recognition. The DCC was preferentially bound upstream of genes, suggesting modulation of transcriptional initiation and polymerase-coupled spreading. Stronger DCC binding upstream of genes with high transcriptional activity indicated a mechanism for tuning DCC activity at specific loci. These data aid in understanding how proteins involved in higher-order chromosome dynamics can regulate transcription at individual loci.

Pre-replication complex organization in the atypical DNA replication cycle of Plasmodium falciparum: characterization of the mini-chromosome maintenance (MCM) complex formation.

The overall organization of cell division in Plasmodium is unique compared to that observed in model organisms because DNA replicates more than once per cell cycle at several points of its life cycle. The sequencing of the Plasmodium genome has also revealed the apparent absence of many key components (e.g. Cdt1, DDK and Cdc45) of the eukaryotic cell cycle machinery that are responsible for the formation of the pre-replication complex (pre-RC). We have characterized the Plasmodium falciparum minichromosome maintenance complex (MCM) that plays a key role in the transition of pre-RC to the RC. Similar to other eukaryotes, the Plasmodium genome encodes six MCM subunits. Here, we show that expression levels of at least three of the PfMCM subunits, the homologues of MCM2, MCM6 and MCM7, change during the intraerythrocytic development cycle, peaking in schizont and decreasing in the ring and trophozoite stages. PfMCM2, 6 and 7 subunits interact with each other to form a developmentally regulated complex: these interactions are detectable in rings and schizonts, but not in trophozoites. PfMCM2, 6 and 7 subunits are localized in both cytosolic and nucleosolic fractions during all intraerythrocytic stages of P. falciparum development, with increased nuclear localization in schizonts. Only PfMCM6 is associated with the chromatin fraction at all stages of growth. No phosphorylation of PfMCM2, 6 and 7 was detected, but two as yet unidentified threonine-phosphosphorylated proteins were present in the complex, whose pattern of phosphorylation varied during parasite development.

PfPK7, an atypical MEK-related protein kinase, reflects the absence of classical three-component MAPK pathways in the human malaria parasite Plasmodium falciparum.

Two members of the mitogen-activated protein kinase (MAPK) family have been previously characterized in Plasmodium falciparum, but in vitro attempts at identifying MAP kinase kinase (MAPKK) homologues have failed. Here we report the characterization of a novel plasmodial protein kinase, PfPK7, whose top scores in blastp analysis belong to the MAPKK3/6 subgroup of MAPKKs. However, homology to MAPKKs is restricted to regions of the C-terminal lobe of the kinase domain, whereas the N-terminal region is closer to fungal protein kinase A enzymes (PKA, members of the AGC group of protein kinases). Hence, PfPK7 is a 'composite' enzyme displaying regions of similarity to more than one protein kinase family, similar to a few other plasmodial protein kinases. PfPK7 is expressed in several developmental stages of the parasite, both in the mosquito vector and in the human host. Recombinant PfPK7 displayed kinase activity towards a variety of substrates, but was unable to phosphorylate the two P. falciparum MAPK homologues in vitro, and was insensitive to PKA and MEK inhibitors. Together with the absence of a typical MAPKK activation site in its T-loop, this suggests that PfPK7 is not a MAPKK orthologue, despite the fact that this enzyme is the most 'MAPKK-like' enzyme encoded in the P. falciparum genome. This is consistent with recent observations that the plasmodial MAPKs are not true orthologues of the ERK1/2, p38 or JNK MAPKs, and strengthens the evidence that classical three-component module-dependent MAPK signalling pathways do not operate in malaria parasites, a feature that has not been described in any other eukaryote.

Identification and initial characterization of three novel cyclin-related proteins of the human malaria parasite Plasmodium falciparum.

The molecular mechanisms regulating cell proliferation and development during the life cycle of malaria parasites remain to be elucidated. The peculiarities of the cell cycle organization during Plasmodium falciparum schizogony suggest that the modalities of cell cycle control in this organism may differ from those in other eukaryotes. Indeed, existing data concerning Plasmodium cell cycle regulators such as cyclin-dependent kinases reveal structural and functional properties that are divergent from those of their homologues in other systems. The work presented here lies in the context of the exploitation of the recently available P. falciparum genome sequence toward the characterization of putative cell cycle regulators. We describe the in silico identification of three open reading frames encoding proteins with maximal homology to various members of the cyclin family and demonstrate that the corresponding polypeptides are expressed in the erythrocytic stages of the infection. We present evidence that these proteins possess cyclin activity by demonstrating either their association with histone H1 kinase activity in parasite extracts or their ability to activate PfPK5, a P. falciparum cyclin-dependent kinase homologue, in vitro. Furthermore, we show that RINGO, a protein with no sequence homology to cyclins but that is nevertheless a strong activator of mammalian CDK1/2, is also a strong activator of PfPK5 in vitro. This raises the possibility that "cryptic" cell cycle regulators may be found among the 50% of the open reading frames in the P. falciparum genome that display no homology to any known proteins.

Molecular characterization and expression of an alternate proliferating cell nuclear antigen homologue, PfPCNA2, in Plasmodium falciparum.

The malaria parasite Plasmodium falciparum genome sequencing has revealed the existence of a second gene for proliferating cell nuclear antigen (PCNA), a key factor in a variety of DNA metabolic events. The alternate copy of PCNA (PfPCNA2) shows only 23% identity to an earlier reported P. falciparum PCNA homologue (PfPCNA1). Our analysis indicated structural conservation of PfPCNA2 compared to eukaryotic PCNAs. PfPCNA1 and 2 polypeptides showed differential expression in the intraerythrocytic cell cycle of the malaria parasite. PfPCNA1 expression slowly increases about threefold from the ring to the late schizont stage. In contrast PfPCNA2 showed robust expression in trophozoites and early schizonts with a sudden drop in expression in the late schizont stage, suggesting that the two PfPCNAs may function under different physiological conditions. Chemical cross-linking indicated the presence of a trimeric PfPCNA2 protein, indicating the possible existence of a functional ring-like PfPCNA2 structure.