Impaired Retromer Function in Niemann-Pick Type C Disease Is Dependent on Intracellular Cholesterol Accumulation.

Niemann-Pick type C disease (NPC) is a rare inherited neurodegenerative disorder characterized by an accumulation of intracellular cholesterol within late endosomes and lysosomes due to NPC1 or NPC2 dysfunction. In this work, we tested the hypothesis that retromer impairment may be involved in the pathogenesis of NPC and may contribute to increased amyloidogenic processing of APP and enhanced BACE1-mediated proteolysis observed in NPC disease. Using NPC1-null cells, primary mouse NPC1-deficient neurons and NPC1-deficient mice (BALB/cNctr-Npc1m1N), we show that retromer function is impaired in NPC. This is manifested by altered transport of the retromer core components Vps26, Vps35 and/or retromer receptor sorLA and by retromer accumulation in neuronal processes, such as within axonal swellings. Changes in retromer distribution in NPC1 mouse brains were observed already at the presymptomatic stage (at 4-weeks of age), indicating that the retromer defect occurs early in the course of NPC disease and may contribute to downstream pathological processes. Furthermore, we show that cholesterol depletion in NPC1-null cells and in NPC1 mouse brains reverts retromer dysfunction, suggesting that retromer impairment in NPC is mechanistically dependent on cholesterol accumulation. Thus, we characterized retromer dysfunction in NPC and propose that the rescue of retromer impairment may represent a novel therapeutic approach against NPC.

The impact of pre-analytical processing on staining quality for H&E, dual hapten, dual color in situ hybridization and fluorescent in situ hybridization assays.

With the advent of personalized medicine, anatomic pathology-based molecular assays, including in situ hybridization (ISH) and mRNA detection tests, are performed routinely in many laboratories and have increased in their clinical importance and complexity. These assays require appropriately fixed tissue samples that preserve both nucleic acid targets and histomorphology to ensure reliable test results for determining patient treatment options. However, all aspects of tissue processing, including time until tissue fixation, type of fixative, duration of fixation, post-fixation treatments, and sectioning of the sample, impact the staining results. ASCO/CAP has issued pre-analytical guidelines to standardize tissue processing for HER2 testing in breast carcinoma specimens: 10% neutral-buffered formalin (NBF) with a fixation time from at least 6 to 48h [1]. Often, this recommendation is not followed to the detriment of staining results [2]. In this paper, we used a human breast carcinoma cell line (MCF7) generated as xenograft tumors as a model system to analyze the effects of different pre-analytical conditions on ISH staining. We performed H&E, FISH and dual colorimetric HER2 ISH assays using specimens fixed across a range of times in six different commonly used fixatives. Additionally, we investigated the effects of varying tissue section thickness, which also impacted the quality of ISH staining. Finally, we evaluated the effects of three different decalcifying solutions on human breast specimens, typically a treatment that occurs post-fixation for evaluating metastases to bone. The results indicate that time and type of fixation treatment, as well as appropriate tissue thickness and post-fixation treatment, all contribute to the quality of ISH staining results. Our data support the ASCO/CAP recommendations for standardized tissue processing (at least 6h in formalin-based fixatives and 4µm section thickness) and indicate that certain fixatives and post-fixative treatments are detrimental to molecular staining results.

Early interrogation and recognition of DNA sequence by indirect readout.

Control of replication, transcription, recombination and repair requires proteins capable of finding particular DNA sequences in a background of a large excess of nonspecific sequences. Such recognition can involve direct readout, with direct contacts to the bases of DNA, or in some cases through the less well-characterized indirect readout mechanisms. In order to measure the relative contributions of direct and indirect readout by a sequence specific endonuclease, HincII, a mutant enzyme deficient in a direct contact, was characterized, and surprisingly showed no loss of sequence specificity. The three dimensional crystal structure shows the loss of most of the direct readout contacts to the DNA, possibly capturing an early stage in target site recognition using predominately indirect readout to prescreen sites before full sequence interrogation.

DNA distortion and specificity in a sequence-specific endonuclease.

Five new structures of the Q138F HincII enzyme bound to a total of three different DNA sequences and three different metal ions (Ca(2+), Mg(2+), and Mn(2+)) are presented. While previous structures were produced from soaking Ca(2+) into preformed Q138F HincII/DNA crystals, the new structures are derived from cocrystallization with Ca(2+), Mg(2+), or Mn(2+). The Mn(2)(+)-bound structure provides the first view of a product complex of Q138F HincII with cleaved DNA. Binding studies and a crystal structure show how Ca(2+) allows trapping of a Q138F HincII complex with noncognate DNA in a catalytically incompetent conformation. Many Q138F HincII/DNA structures show asymmetry, despite the binding of a symmetric substrate by a symmetric enzyme. The various complexes are fit into a model describing the different conformations of the DNA-bound enzyme and show how DNA conformational energetics determine DNA-cleavage rates by the Q138F HincII enzyme.

Cooperative DNA binding by CI repressor is dispensable in a phage lambda variant.

Complex gene regulatory circuits contain many interacting components. In principle, all of these components and interactions may be essential to the function of the circuit. Alternatively, some of them may be refinements to a simpler version of the circuit that improve its fitness. In this work, we have tested whether a particular property of a critical regulatory protein, CI, is essential to the behavior of the phage lambda regulatory circuit. In the lysogenic state, CI represses the expression of the lytic genes, allowing a stable lysogenic state, by binding cooperatively to six operators. A mutant phage lacking cooperativity because of a change in cI could not form stable lysogens; however, this defect could be suppressed by the addition of mutations that altered two cis-acting sites but did not restore cooperativity. The resulting triple mutant was able to grow lytically, form stable single lysogens, and switch to lytic growth upon prophage induction, showing a threshold response in switching similar to that of wild-type lambda. We conclude that cooperative DNA binding by CI is not essential for these properties of the lambda circuitry, provided that suppressors increase the level of CI. Unlike wild-type lysogens, mutant lysogens were somewhat unstable under certain growth conditions. We surmise that cooperativity is a refinement to a more basic circuit, and that it affords increased stability to the lysogenic state in response to environmental variations.

Conservation and diversity in the immunity regions of wild phages with the immunity specificity of phage lambda.

The gene regulatory circuitry of phage lambda is among the best-understood circuits. Much of the circuitry centres around the immunity region, which includes genes for two repressors, CI and Cro, and their cis-acting sites. Related phages, termed lambdoid phages, have different immunity regions, but similar regulatory circuitry and genome organization to that of lambda, and show a mosaic organization, arising by recombination between lambdoid phages. We sequenced the immunity regions of several wild phages with the immunity specificity of lambda, both to determine whether natural variation exists in regulation, and to analyse conservation and variability in a region rich in well-studied regulatory elements. CI, Cro and their cis-acting sites are almost identical to those in lambda, implying that regulatory mechanisms controlled by the immunity region are conserved. A segment adjacent to one of the operator regions is also conserved, and may be a novel regulatory element. In most isolates, different alleles of two regulatory proteins (N and CII) flank the immunity region; possibly the lysis-lysogeny decision is more variable among isolates. Extensive mosaicism was observed for several elements flanking the immunity region. Very short sequence elements or microhomologies were also identified. Our findings suggest mechanisms by which fine-scale mosaicism arises.