Biophysical characteristics of lipid-induced Aß oligomers correlate to distinctive phenotypes in transgenic mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects cognition and memory. Recent advances have helped identify many clinical sub-types in AD. Mounting evidence point toward structural polymorphism among fibrillar aggregates of amyloid-ß (Aß) to being responsible for the phenotypes and clinical manifestations. In the emerging paradigm of polymorphism and prion-like propagation of aggregates in AD, the role of low molecular weight soluble oligomers, which are long known to be the primary toxic agents, in effecting phenotypes remains inconspicuous. In this study, we present the characterization of three soluble oligomers of Aß42, namely 14LPOs, 16LPOs, and GM1Os with discreet biophysical and biochemical properties generated using lysophosphatidyl glycerols and GM1 gangliosides. The results indicate that the oligomers share some biophysical similarities but display distinctive differences with GM1Os. Unlike the other two, GM1Os were observed to be complexed with the lipid upon isolation. It also differs mainly in detection by conformation-sensitive dyes and conformation-specific antibodies, temperature and enzymatic stability, and in the ability to propagate morphologically-distinct fibrils. GM1Os also show distinguishable biochemical behavior with pronounced neuronal toxicity. Furthermore, all the oligomers induce cerebral amyloid angiopathy (CAA) and plaque burden in transgenic AD mice, which seems to be a consistent feature among all lipid-derived oligomers, but 16LPOs and GM1Os displayed significantly higher effect than the others. These results establish a correlation between molecular features of Aß42 oligomers and their distinguishable effects in transgenic AD mice attuned by lipid characteristics, and therefore help bridge the knowledge gap in understanding how oligomer conformers could elicit AD phenotypes.

Congenic mice demonstrate the presence of QTLs conferring obesity and hypercholesterolemia on chromosome 1 in the TALLYHO mouse.

The TALLYHO (TH) mouse presents a metabolic syndrome of obesity, type 2 diabetes, and hyperlipidemia. Highly significant quantitative trait loci (QTLs) linked to adiposity and hypercholesterolemia were previously identified on chromosome (Chr) 1 in a genome-wide scan of F2 mice from C57BL/6J (B6) x TH. In this study, we generated congenic mouse strains that carry the Chr 1 QTLs derived from TH on a B6 background; B6.TH-Chr1-128Mb (128Mb in size) and B6.TH-Chr1-92Mb (92Mb in size, proximally overlapping). We characterized these congenic mice on chow and high fat (HF) diets. On chow, B6.TH-Chr1-128Mb congenic mice exhibited a slightly larger body fat mass compared with B6.TH-Chr1-92Mb congenic and B6 mice, while body fat mass between B6.TH-Chr1-92Mb congenic and B6 mice was comparable. Plasma total cholesterol levels were significantly higher in B6.TH-Chr1-128Mb congenics compared to B6.TH-Chr1-92Mb congenic and B6 mice. Again, there was no difference in plasma total cholesterol levels between B6.TH-Chr1-92Mb congenic and B6 mice. All animals gained more body fat and exhibited higher plasma total cholesterol levels when fed HF diets than fed chow, but these increases were greater in B6.TH-Chr1-128Mb congenics than in B6.TH-Chr1-92Mb congenic and B6 mice. These results confirmed the effect of the 128Mb TH segment from Chr 1 on body fat and plasma cholesterol values and showed that the distal segment of Chr 1 from TH is necessary to cause both phenotypes. Through bioinformatic approaches, we generated a list of potential candidate genes within the distal region of Chr 1 and tested Ifi202b and Apoa2. We conclude that Chr 1 QTLs largely confer obesity and hypercholesterolemia in TH mice and can be promising targets for identifying susceptibility genes. Congenic mouse strains will be a valuable resource for gene identification.

Multiple Factors Influence the Incubation Period of ALS Prion-like Transmission in SOD1 Transgenic Mice.

Mutations in superoxide dismutase 1 (SOD1) that are associated with amyotrophic lateral sclerosis (ALS) cause its misfolding and aggregation. Prior studies have demonstrated that the misfolded conformation of ALS-SOD1 can template with naïve SOD1 "host proteins" to propagate, spread, and induce paralysis in SOD1 transgenic mice. These observations have advanced the argument that SOD1 is a host protein for an ALS conformer that is prion-like and experimentally transmissible. Here, we investigated the propagation of different isolates of G93A-SOD1 ALS conformers using a paradigm involving transmission to mice expressing human G85R-SOD1 fused to yellow fluorescent protein (G85R-SOD1:YFP). In these studies, we also utilized a newly developed line of mice in which the G85R-SOD1:YFP construct was flanked by loxp sites, allowing its temporal and spatial regulation. We used methods in which the G93A ALS conformers were injected into the sciatic nerve or hindlimb muscle of adult transgenic mice. We observed that the incubation period to paralysis varied significantly depending upon the source of inoculum containing misfolded G93A SOD1. Serial passage and selection produced stable isolates of G93A ALS conformers that exhibited a defined minimum incubation period of ~2.5 months when injected into the sciatic nerve of young adult mice. As expected, neuronal excision of the transgene in loxpG85R-SOD1:YFP mice blocked induction of paralysis by transmission of G93A ALS conformers. Our findings indicate that G93A ALS conformers capable of inducing disease require neuronal expression of a receptive host SOD1 protein for propagation, with a defined incubation period to paralysis.

A C1qTNF3 collagen domain fusion chaperones diverse secreted proteins and anti-Aß scFvs: Applications for gene therapies.

Enhancing production of protein cargoes delivered by gene therapies can improve efficacy by reducing the amount of vector or simply increasing transgene expression levels. We explored the utility of a 126-amino acid collagen domain (CD) derived from the C1qTNF3 protein as a fusion partner to chaperone secreted proteins, extracellular "decoy receptor" domains, and single-chain variable fragments (scFvs). Fusions to the CD domain result in multimerization and enhanced levels of secretion of numerous fusion proteins while maintaining functionality. Efficient creation of bifunctional proteins using the CD domain is also demonstrated. Recombinant adeno-associated viral vector delivery of the CD with a signal peptide resulted in high-level expression with minimal biological impact as assessed by whole-brain transcriptomics. As a proof-of-concept in vivo study, we evaluated three different anti-amyloid Aß scFvs (anti-Aß scFvs), alone or expressed as CD fusions, following viral delivery to neonatal CRND8 mice. The CD fusion increased half-life, expression levels, and improved efficacy for amyloid lowering of a weaker binding anti-Aß scFv. These studies validate the potential utility of this small CD as a fusion partner for secretory cargoes delivered by gene therapy and demonstrate that it is feasible to use this CD fusion to create biotherapeutic molecules with enhanced avidity or bifunctionality.

Aß Amyloid Scaffolds the Accumulation of Matrisome and Additional Proteins in Alzheimer's Disease.

We report a highly significant correlation in brain proteome changes between Alzheimers disease (AD) and CRND8 APP695NL/F transgenic mice. However, integrating protein changes observed in the CRND8 mice with co-expression networks derived from human AD, reveals both conserved and divergent module changes. For the most highly conserved module (M42, matrisome) we find many proteins accumulate in plaques, cerebrovascular amyloid (CAA), dystrophic processes, or a combination thereof. Overexpression of two M42 proteins, midkine (Mdk) and pleiotrophin (PTN), in CRND8 mice brains leads to increased accumulation of A ß ; in plaques and in CAA; further, recombinant MDK and PTN enhance A ß ; aggregation into amyloid. Multiple M42 proteins, annotated as heparan sulfate binding proteins, bind to fibrillar A ß 42 and a non-human amyloid fibril in vitro. Supporting this binding data, MDK and PTN co-accumulate with transthyretin (TTR) amyloid in the heart and islet amyloid polypeptide (IAPP) amyloid in the pancreas. Our findings establish several critical insights. Proteomic changes in modules observed in human AD brains define an A ß ; amyloid responsome that is well conserved from mouse model to human. Further, distinct amyloid structures may serve as scaffolds, facilitating the co-accumulation of proteins with signaling functions. We hypothesize that this co-accumulation may contribute to downstream pathological sequalae. Overall, this contextualized understanding of proteomic changes and their interplay with amyloid deposition provides valuable insights into the complexity of AD pathogenesis and potential biomarkers and therapeutic targets.

Absolute lymphocyte count is associated with survival in ovarian cancer independent of tumor-infiltrating lymphocytes.

BACKGROUND: The immune system strongly influences outcome in patients with ovarian cancer. In particular, the absolute lymphocyte count in peripheral blood (ALC) and the presence of tumor-infiltrating lymphocytes (TIL) have each been associated with favourable prognosis. However, the mechanistic relationships between ALC, TIL and prognosis are poorly understood. We hypothesized that high ALC values might be associated with stronger tumor immunity as manifested by increased TIL, decreased tumor burden and longer survival. METHODS: ALC values were collected from patient records ≥ 2 years before, during or after primary treatment for high-grade serous ovarian cancer (HGSC). Lymphocyte subsets were assessed in peripheral blood by flow cytometry. CD8+ and CD20+ TIL were assessed by immunohistochemistry. RESULTS: Overall, patients had normal ALC values two or more years prior to diagnosis of HGSC. These values were not predictive of disease severity or survival upon subsequent development of HGSC. Rather, ALC declined upon development of HGSC in proportion to disease burden. This decline involved all lymphocyte subsets. ALC increased following surgery, remained stable during chemotherapy, but rarely recovered to pre-diagnostic levels. ALC values recorded at diagnosis did not correlate with CD8+ or CD20+ TIL but were associated with progression-free survival. CONCLUSIONS: Patients with high intrinsic ALC values show no clinical or survival advantage upon subsequent development of HGSC. ALC values at diagnosis are prognostic due to an association with disease burden rather than TIL. Therapeutic enhancement of ALC may be necessary but not sufficient to improve survival in HGSC.

Subcongenic analysis of tabw2 obesity QTL on mouse chromosome 6.

BACKGROUND: We previously established a congenic mouse strain with TALLYHO/Jng (TH) donor segment on chromosome 6 in a C57BL/6 (B6) background that harbors an obesity quantitative trait locus, tabw2. The B6.TH-tabw2 congenic mice developed increased adiposity that became exacerbated upon feeding a high fat-high sucrose (HFS) diet. To fine map the tabw2, in this study we generated and characterized subcongenic lines with smaller TH donor segments. RESULTS: We fixed four subcongenic lines, with maximum size of donor segment retained in the lines ranging from 10.8 - 92.5 Mb. For mapping, all the subcongenic mice, along with B6.TH-tabw2 congenic and B6-homozygous control mice were fed either chow or HFS diets, and their post-mortem fat pads were weighed. Mice were also characterized for energy expenditure, respiratory exchange ratio, locomotor activity, and food intake. As previously reported, B6.TH-tabw2 congenic mice showed a significantly larger fat mass than controls on both diets. On chow, a subcongenic line retaining the distal region of the TH donor congenic interval exhibited significantly larger fat mass than B6-homozygous controls, and comparable that to B6.TH-tabw2 congenic mice. Two nested subcongenic lines within that region suggested that the effect of tabw2 on obesity could be attributed to at least two subloci. On HFS diets, on the other hand, all the subcongenic mice had significantly larger fat mass than controls without genotype differences, but none of them had fat mass as large as the original congenic mice. This possibly implicates that further genetic complexity involves in the effect of tabw2 on diet-induced obesity. Significantly reduced locomotor activity was exhibited in B6.TH-tabw2 congenic and subcongenic mice compared to controls when animals were fed HFS diets. B6.TH-tabw2 congenic mice, but not subcongenic mice, also had significantly increased food intake on HFS diets. CONCLUSIONS: It appears that at least two subloci explaining the tabw2 effect under chow feeding map to the distal region of the congenic interval, whereas the diet-induced obesity mediated by tabw2 is attributed to more complex genetic mechanism.

Variation in the vulnerability of mice expressing human superoxide dismutase 1 to prion-like seeding: a study of the influence of primary amino acid sequence.

Misfolded forms of superoxide dismutase 1 (SOD1) with mutations associated with familial amyotrophic lateral sclerosis (fALS) exhibit prion characteristics, including the ability to act as seeds to accelerate motor neuron disease in mouse models. A key feature of infectious prion seeding is that the efficiency of transmission is governed by the primary sequence of prion protein (PrP). Isologous seeding, where the sequence of the PrP in the seed matches that of the host, is generally much more efficient than when there is a sequence mis-match. Here, we used paradigms in which mutant SOD1 seeding homogenates were injected intraspinally in newborn mice or into the sciatic nerve of adult mice, to assess the influence of SOD1 primary sequence on seeding efficiency. We observed a spectrum of seeding efficiencies depending upon both the SOD1 expressed by mice injected with seeds and the origin of the seed preparations. Mice expressing WT human SOD1 or the disease variant G37R were resistant to isologous seeding. Mice expressing G93A SOD1 were also largely resistant to isologous seeding, with limited success in one line of mice that express at low levels. By contrast, mice expressing human G85R-SOD1 were highly susceptible to isologous seeding but resistant to heterologous seeding by homogenates from paralyzed mice over-expressing mouse SOD1-G86R. In other seeding experiments with G85R SOD1:YFP mice, we observed that homogenates from paralyzed animals expressing the H46R or G37R variants of human SOD1 were less effective than seeds prepared from mice expressing the human G93A variant. These sequence mis-match effects were less pronounced when we used purified recombinant SOD1 that had been fibrilized in vitro as the seeding preparation. Collectively, our findings demonstrate diversity in the abilities of ALS variants of SOD1 to initiate or sustain prion-like propagation of misfolded conformations that produce motor neuron disease.

Aß40 displays amyloidogenic properties in the non-transgenic mouse brain but does not exacerbate Aß42 toxicity in Drosophila.

BACKGROUND: Self-assembly of the amyloid-ß (Aß) peptide into aggregates, from small oligomers to amyloid fibrils, is fundamentally linked with Alzheimer's disease (AD). However, it is clear that not all forms of Aß are equally harmful and that linking a specific aggregate to toxicity also depends on the assays and model systems used (Haass et al., J Biol. Chem 269:17741-17748, 1994; Borchelt et al., Neuron 17:1005-1013, 1996). Though a central postulate of the amyloid cascade hypothesis, there remain many gaps in our understanding regarding the links between Aß deposition and neurodegeneration. METHODS: In this study, we examined familial mutations of Aß that increase aggregation and oligomerization, E22G and ΔE22, and induce cerebral amyloid angiopathy, E22Q and D23N. We also investigated synthetic mutations that stabilize dimerization, S26C, and a phospho-mimetic, S8E, and non-phospho-mimetic, S8A. To that end, we utilized BRI2-Aß fusion technology and rAAV2/1-based somatic brain transgenesis in mice to selectively express individual mutant Aß species in vivo. In parallel, we generated PhiC31-based transgenic Drosophila melanogaster expressing wild-type (WT) and Aß40 and Aß42 mutants, fused to the Argos signal peptide to assess the extent of Aß42-induced toxicity as well as to interrogate the combined effect of different Aß40 and Aß42 species. RESULTS: When expressed in the mouse brain for 6 months, Aß42 E22G, Aß42 E22Q/D23N, and Aß42WT formed amyloid aggregates consisting of some diffuse material as well as cored plaques, whereas other mutants formed predominantly diffuse amyloid deposits. Moreover, while Aß40WT showed no distinctive phenotype, Aß40 E22G and E22Q/D23N formed unique aggregates that accumulated in mouse brains. This is the first evidence that mutant Aß40 overexpression leads to deposition under certain conditions. Interestingly, we found that mutant Aß42 E22G, E22Q, and S26C, but not Aß40, were toxic to the eye of Drosophila. In contrast, flies expressing a copy of Aß40 (WT or mutants), in addition to Aß42WT, showed improved phenotypes, suggesting possible protective qualities for Aß40. CONCLUSIONS: These studies suggest that while some Aß40 mutants form unique amyloid aggregates in mouse brains, they do not exacerbate Aß42 toxicity in Drosophila, which highlights the significance of using different systems for a better understanding of AD pathogenicity and more accurate screening for new potential therapies.

Psychotherapy and pharmacotherapy for patients with dissociative identity disorder.

There is a wide variety of what have been called "dissociative disorders," including dissociative amnesia, dissociative fugue, depersonalization disorder, dissociative identity disorder, and forms of dissociative disorder not otherwise specified. Some of these diagnoses, particularly dissociative identity disorder, are controversial and have been questioned by many clinicians over the years. The disorders may be under-diagnosed or misdiagnosed, but many persons who have experienced trauma report "dissociative" symptoms. Prevalence of dissociative disorders is unknown, but current estimates are higher than previously thought. This paper reviews clinical, phenomenological, and epidemiological data regarding diagnosis in general, and illustrates possible treatment interventions for dissociative identity disorder, with a focus on psychotherapy interventions and a review of current psychopharmacology recommendations as part of a comprehensive multidisciplinary treatment plan.

Islet Insulin Secretion, β-Cell Mass, and Energy Balance in a Polygenic Mouse Model of Type 2 Diabetes With Obesity

Abstract Type 2 diabetes (T2D) and obesity are polygenic metabolic diseases, highly prevalent in humans. The TALLYHO/Jng (TH) mouse is a polygenic model of T2D and obesity that encompasses many aspects of the human conditions. In this study, we investigated the key metabolic components including β-cell physiology and energy balance involved in the development of diabetes and obesity in TH mice. Glucose-stimulated insulin secretion from freshly isolated islets was significantly enhanced in TH mice compared with normal C57BL/6 (B6) mice, similar to the compensated stage in human T2D associated with obesity. This increased glucose responsiveness was accompanied by an increase in total β-cell mass in TH mice. Energy expenditure and locomotor activity were significantly reduced in TH mice compared with B6 mice. Food intake was comparable between the two strains but water intake was more in TH mice. Together, obesity in TH mice does not appear to be due to hyperphagia, and TH mice may be a genetic model for T2D with obesity, allowing study of the important signaling or metabolic pathways leading to compensatory increases in insulin secretion and β-cell mass in insulin resistance.