A key adaptation enabling the fetus to survive in a limited energy environment may be the reprogramming of mitochondrial function, which can have deleterious effects. Critical questions are whether mitochondrial dysfunction progressively declines after birth, and if so, what mechanism might underlie this process. To address this, we developed a model of intrauterine growth retardation (IUGR) in the rat that leads to diabetes in adulthood. Reactive oxygen species (ROS) production and oxidative stress gradually increased in IUGR islets. ATP production was impaired and continued to deteriorate with age. The activities of complex I and III of the electron transport chain progressively declined in IUGR islets. Mitochondrial DNA point mutations accumulated with age and were associated with decreased mitochondrial DNA content and reduced expression of mitochondria-encoded genes in IUGR islets. Mitochondrial dysfunction resulted in impaired insulin secretion. These results demonstrate that IUGR induces mitochondrial dysfunction in the fetal ␤-cell, leading to increased production of ROS, which in turn damage mitochondrial DNA. A self-reinforcing cycle of progressive deterioration in mitochondrial function leads to a corresponding decline in ␤-cell function. Finally, a threshold in mitochondrial dysfunction and ROS production is reached, and diabetes ensues.Epidemiological studies have revealed strong statistical links between poor fetal growth and the subsequent development of type 2 diabetes in adulthood (1-6). These associations have led to the "fetal origins of adult disease hypothesis," which states that "to ensure fetal survival, adaptations to intrauterine deprivation result in a permanent reprogramming of key organ systems" (2). Both insulin action and insulin secretion are impaired in individuals who were growth-retarded at birth (7-12).Uteroplacental insufficiency, the most common cause of in utero growth retardation, limits the supply of critical substrates such as oxygen, glucose, and amino acids to the fetus and results in poor fetal growth (13)(14)(15). This abnormal metabolic intrauterine milieu affects development of the fetus by modifying gene expression and function of susceptible cells, such as the ␤-cell (16 -19). The molecular mechanisms responsible for permanent changes in gene expression are not known, but they are a critical element in our understanding of how factors in early development can lead to long term consequences in aging and disease.A key adaptation enabling the fetus to survive in a limited energy environment may be the reprogramming of mitochondrial function (17, 18). However, these alterations in mitochondrial function can have deleterious effects, especially in cells that have a high energy requirement, such as the ␤-cell. The ␤-cell depends upon the normal production of ATP for nutrientinduced insulin secretion (20 -27) and proliferation (28). Thus, an interruption of mitochondrial function can have profound consequences for the ␤-cell.Mitochondrial dysfunction can also lead to inc...

The circulating antibody repertoire encodes a patient's health status and pathogen exposure history, but identifying antibodies with diagnostic potential usually requires knowledge of the antigen(s). We previously circumvented this problem by screening libraries of bead-displayed small molecules against case and control serum samples to discover “epitope surrogates” (ligands of IgGs enriched in the case sample). Here, we describe an improved version of this technology that employs DNA-encoded libraries and high-throughput FACS-based screening to discover epitope surrogates that differentiate noninfectious/latent (LTB) patients from infectious/active TB (ATB) patients, which is imperative for proper treatment selection and antibiotic stewardship. Normal control/LTB (10 patients each, NCL) and ATB (10 patients) serum pools were screened against a library (5 × 106 beads, 448k unique compounds) using fluorescent anti-human IgG to label hit compound beads for FACS. Deep sequencing decoded all hit structures and each hit's occurrence frequencies. ATB hits were pruned of NCL hits and prioritized for resynthesis based on occurrence and homology. Several structurally homologous families were identified and 16/21 resynthesized representative hits validated as selective ligands of ATB serum IgGs (p < 0.005). The native secreted TB protein Ag85B (though not the E. coli recombinant form) competed with one of the validated ligands for binding to antibodies, suggesting that it mimics a native Ag85B epitope. The use of DNA-encoded libraries and FACS-based screening in epitope surrogate discovery reveals thousands of potential hit structures. Distilling this list down to several consensus chemical structures yielded a diagnostic panel for ATB composed of thermally stable and economically produced small molecule ligands in place of protein antigens.

Prions consist mainly of PrP Sc , a pathogenic conformer of hostencoded PrP C . Prion populations with distinct phenotypes but associated with PrP Sc , having the same amino acid sequence, constitute distinct strains. Strain identity is thought to be encoded by the conformation of PrP Sc and to be maintained by seeded conversion. Prion strains can be distinguished by the cell panel assay, which measures their ability to infect distinct cell lines. Brainderived 22L prions characteristically are able to infect R33 cells (i.e., are "R33 competent"), as well as PK1 cells in the presence of the inhibitor swainsonine (i.e. are "swa resistant"). Here we report that 22L prions retained their characteristic cell tropism and swa resistance when transferred from brain to R33 cells. However, when transferred from the R33 cells to PK1 cells, they gradually became R33 incompetent and swa sensitive, unless the transfer was in the presence of swa, in which case swa resistance and R33 competence were retained. PrP Sc associated with swaresistant/R33-competent and swa-sensitive/R33-incompetent prions had different conformational stabilities. When cloned R33-incompetent/swa-sensitive prions were again propagated in brain, their properties gradually reverted to those of the original brain-derived 22L prions. Our results support the view that 22L prion populations are heterogeneous and that distinct prion variants are selected in different cellular environments.evolution | scrapie cell assay | selection W e determine the susceptibility of a cell line to a prion strain by the standard scrapie cell assay (SSCA) (1, 2). In short, we expose cells to various dilutions of the prion sample, propagate them for three splits, and determine the proportion of PrP Sc -containing cells by ELISA. We define as response index (RI) the reciprocal of the dilution that yields a designated proportion of infected cells under standard conditions (usually 300 PrP Sc -positive cells/20,000 cells).We discriminate murine prion strains by the cell panel assay (CPA) (3), which is based on the differential susceptibility of selected cell lines to individual prion strains. RML, 22L, ME7, and 301C prions can be distinguished by their relative RI values on a panel consisting of neuroblastoma-derived PK1 cells in the presence or absence of swainsonine (swa), neuroblastoma-derived R33 (or, recently, the more susceptible subclone R33 2H11 ) cells, fibroblastic LD9 and CNS-derived CAD cells. Swa, an inhibitor of complex glycosylation (4), suppresses infection of PK1 cells by RML but not by 22L prions (5).As recently reported, when 22L prions were transferred from brain to cultured PK1 cells, the PK1 cell-adapted 22L variants gradually outgrew the original population, as documented by a profound change in their CPA characteristics: Although brainderived 22L prions efficiently infected R33 cells (i.e., were R33 competent) and PK1 cells in the presence of swa (i.e., were swa resistant), the PK1 cell-adapted 22L prions were R33 incompetent (i.e., were 1.5-2 logs less infec...

PrPC, a host protein which in prion-infected animals is converted to PrPSc, is linked to the cell membrane by a GPI anchor. Mice expressing PrPC without GPI anchor (tgGPI- mice), are susceptible to prion infection but accumulate anchorless PrPSc extra-, rather than intracellularly. We investigated whether tgGPI− mice could faithfully propagate prion strains despite the deviant structure and location of anchorless PrPSc. We found that RML and ME7, but not 22L prions propagated in tgGPI− brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA). Surprisingly, the levels of proteinase K-resistant PrPSc (PrPres) in RML- or ME7-infected tgGPI− brain were 25–50 times higher than in wild-type brain. When returned to wild-type brain, ME7 prions recovered their original properties, however RML prions had given rise to a novel prion strain, designated SFL, which remained unchanged even after three passages in wild-type mice. Because both RML PrPSc and SFL PrPSc are stably propagated in wild-type mice we propose that the two conformations are separated by a high activation energy barrier which is abrogated in tgGPI− mice.

No abstract

The activity-dependent plasticity of synapses is believed to be the cellular basis of learning. These synaptic changes are mediated through the coordination of local biochemical reactions in synapses and changes in gene transcription in the nucleus to modulate neuronal circuits and behavior. The protein kinase C (PKC) family of isozymes has long been established as critical for synaptic plasticity. However, due to a lack of suitable isozyme-specific tools, the role of the novel subfamily of PKC isozymes is largely unknown. Here, through the development of FLIM-FRET activity sensors, we investigate novel PKC isozymes in synaptic plasticity in CA1 pyramidal neurons of mice of either sex. We find that PKCδ is activated downstream of TrkB and DAG production and that the spatiotemporal nature of its activation depends on the plasticity stimulation. In response to single-spine plasticity, PKCδ is activated primarily in the stimulated spine and is required for local expression of plasticity. However, in response to multi-spine stimulation, a long-lasting and spreading activation of PKCδ scales with the number of spines stimulated and, by regulating CREB activity, couples spine plasticity to transcription in the nucleus. Thus, PKCδ plays a dual functional role in facilitating synaptic plasticity.SIGNIFICANCE STATEMENT:Synaptic plasticity, or the ability to change the strength of the connections between neurons, underlies learning and memory and is critical for brain health. The protein kinase C (PKC) family is central to this process. However, understanding how these kinases work to mediate plasticity has been limited by a lack of tools to visualize and perturb their activity. Here, we introduce and use new tools to reveal a dual role for PKCδ in facilitating local synaptic plasticity and stabilizing this plasticity through spine-to-nucleus signaling to regulate transcription. This work provides new tools to overcome limitations in studying isozyme-specific PKC function and provides insight into molecular mechanisms of synaptic plasticity.

Rab-dependent membrane trafficking is critical for changing the structure and function of dendritic spines during synaptic plasticity. Here, we developed highly sensitive sensors to monitor Rab protein activity in single dendritic spines undergoing structural long-term potentiation (sLTP) in rodent organotypic hippocampal slices. During sLTP, Rab10 was persistently inactivated (>30 min) in the stimulated spines, whereas Rab4 was transiently activated over ~5 min. Inhibiting or deleting Rab10 enhanced sLTP, electrophysiological LTP and AMPA receptor (AMPAR) insertion during sLTP. In contrast, disrupting Rab4 impaired sLTP only in the first few minutes, and decreased AMPAR insertion during sLTP. Thus, our results suggest that Rab10 and Rab4 oppositely regulate AMPAR trafficking during sLTP, and disinhibition of Rab10 signaling gates the induction of LTP and associated spine structural plasticity.

Rab-dependent membrane trafficking is critical for changing the structure and function of dendritic spines during synaptic plasticity. Here, we developed highly sensitive sensors to monitor Rab protein activity in single dendritic spines undergoing structural long-term potentiation (sLTP) in rodent organotypic hippocampal slices. During sLTP, Rab10 was persistently inactivated (>30 min) in the stimulated spines, whereas Rab4 was transiently activated over ~5 min. Inhibiting or deleting Rab10 enhanced sLTP, electrophysiological LTP and AMPA receptor (AMPAR) insertion during sLTP. In contrast, disrupting Rab4 impaired sLTP only in the first few minutes, and decreased AMPAR insertion during sLTP. Thus, our results suggest that Rab10 and Rab4 oppositely regulate AMPAR trafficking during sLTP, and disinhibition of Rab10 signaling gates the induction of LTP and associated spine structural plasticity.