Parenchymal Deposition Research Articles

Cryo-EM structures of Aβ40 filaments from the leptomeninges of individuals with Alzheimer’s disease and cerebral amyloid angiopathy

We used electron cryo-microscopy (cryo-EM) to determine the structures of Aβ40 filaments from the leptomeninges of individuals with Alzheimer’s disease and cerebral amyloid angiopathy. In agreement with previously reported structures, which were solved to a resolution of 4.4 Å, we found three types of filaments. However, our new structures, solved to a resolution of 2.4 Å, revealed differences in the sequence assignment that redefine the fold of Aβ40 peptides and their interactions. Filaments are made of pairs of protofilaments, the ordered core of which comprises D1–G38. The different filament types comprise one, two or three protofilament pairs. In each pair, residues H14–G37 of both protofilaments adopt an extended conformation and pack against each other in an anti-parallel fashion, held together by hydrophobic interactions and hydrogen bonds between main chains and side chains. Residues D1–H13 fold back on the adjacent parts of their own chains through both polar and non-polar interactions. There are also several additional densities of unknown identity. Sarkosyl extraction and aqueous extraction gave the same structures. By cryo-EM, parenchymal deposits of Aβ42 and blood vessel deposits of Aβ40 have distinct structures, supporting the view that Alzheimer’s disease and cerebral amyloid angiopathy are different Aβ proteinopathies.

Distinct Molecular Signatures of Amyloid-Beta and Tau in Alzheimer's Disease Associated with Down Syndrome.

Limited comparative data exist on the molecular spectrum of amyloid-beta (Aβ) and tau deposition in individuals with Down syndrome (DS) and sporadic Alzheimer's disease (sAD). We assessed Aβ and tau deposition severity in the temporal lobe and cerebellum of ten DS and ten sAD cases. Immunohistochemistry was performed using antibodies against eight different Aβ epitopes (6F/3D, Aβ38, Aβ39, Aβ40, Aβ42, Aβ43, pyroglutamate Aβ at third glutamic acid (AβNp3E), phosphorylated- (p-)Aβ at 8th serine (AβpSer8)), and six different pathological tau epitopes (p-Ser202/Thr205, p-Thr231, p-Ser396, Alz50, MC1, GT38). Findings were evaluated semi-quantitatively and quantitatively using digital pathology. DS cases had significantly higher neocortical parenchymal deposition (Aβ38, Aβ42, and AβpSer8), and cerebellar parenchymal deposition (Aβ40, Aβ42, AβNp3E, and AβpSer8) than sAD cases. Furthermore, DS cases had a significantly larger mean plaque size (6F/3D, Aβ42, AβNp3E) in the temporal lobe, and significantly greater deposition of cerebral and cerebellar Aβ42 than sAD cases in the quantitative analysis. Western blotting corroborated these findings. Regarding tau pathology, DS cases had significantly more severe cerebral tau deposition than sAD cases, especially in the white matter (p-Ser202/Thr205, p-Thr231, Alz50, and MC1). Greater total tau deposition in the white matter (p-Ser202/Thr205, p-Thr231, and Alz50) of DS cases was confirmed by quantitative analysis. Our data suggest that the Aβ and tau molecular signatures in DS are distinct from those in sAD.

Neuropathology of central nervous system involvement in TTR amyloidosis

Hereditary transthyretin amyloidosis (ATTRv) is a systemic disease caused by the accumulation of misfolded transthyretin (TTR). It usually presents with an adult-onset progressive axonal peripheral neuropathy and cardiomyopathy. In the central nervous system (CNS), variant TTR is produced by the choroid plexus and accumulates in the leptomeninges. CNS symptoms have been increasingly recognized in this population, including transient focal neurological episodes and stroke, particularly in patients with the V30M mutation and longstanding disease. The prevalence, pathophysiology, and progression of CNS involvement remain to be clarified. The present work explores if there is a recognizable sequence of CNS TTR deposition in ATTRv. We studied the topographical and severity distribution of TTR deposition in 16 patients with ATTRv, aged 27–69 years and with a mean disease duration of 10.9 years (range: 3–29). Our results suggest that CNS pathological involvement in V30M ATTRv occurs early in the disease course, probably starting in pre-symptomatic phases, and follows a distinct sequence. Leptomeninges and subarachnoid meningeal vessels are affected earlier, then followed by perforating cortical vessels and subpial deposition, and finally by deposition in the subependymal and basal ganglia vessels near the ependymal lining. Brainstem and spinal cord show early and severe involvement, with amyloid subpial deposition already seen in initial stages. Despite massive superficial amyloid deposition, no parenchymal deposition outside subpial or subependymal regions was found. Additionally, vascular lesions or superficial cortical siderosis were not frequent. Future studies with more patients from different populations and TTR mutations will be important to confirm these findings. Defining stages of TTR pathology in the CNS may be useful to better understand pathogenic mechanisms leading to symptoms and to interpret neuroimaging biomarkers.

IMMU-16. Next-generation CAR T-cells targeting IL13RA2 and secreting IL-15 achieve durable tumour clearance in pre-clinical model of diffuse midline glioma

T-cells engineered with a chimeric antigen receptor (CAR) to acquire tumour specificity provide a possible new treatment approach for childhood brain tumours. Durable clinical remissions have been achieved with CD19-directed CAR T-cells in refractory B-cell malignancies including control of leptomeningeal disease and parenchymal deposits. Early clinical data of CAR-T cells in adult glioblastoma and diffuse midline glioma (DMG) further support the rationale for development of CAR-T cell therapy for paediatric high-grade gliomas (pHGG) and other high-risk childhood brain tumours. IL13RA2 provides a suitable CAR target. It is expressed in the majority of pHGG including DMGs while expression is absent on normal (paediatric) brain tissue. Early results with CAR T-cell therapy for adult HGG has shown that IL13RA2 can be safely targeted, and potent anti-tumour activity is possible. However, responses are variable and ultimately transient. This is likely due to the immunosuppressive environment encountered by CAR T-cells at tumour sites which hampers persistent tumour-directed immunity. Here we develop a next generation CAR approach engineering T-cells with both a CAR and a cytokine signal which supports persistent anti-tumour immunity. We have generated novel IL13RA2-specific antibodies and used these to construct CARs. Using a functional screening approach assessing target-specific cytolytic function, T-cell proliferation and cytokine release, we selected the optimal IL13RA2-CAR design. Then, we generated a next generation CAR construct co-expressing inflammatory cytokine IL-15 with the IL13RA2-CAR. In a PDX model of DMG, T-cells transduced with IL13RA2-CARIL-15, in contrast to IL13RA2-CAR alone, induced long-term tumour clearance. Further in vitro testing of IL13RA2-CARIL-15 showed enhanced proliferation and resistance to immune-suppressive secreted factors such as TGF-β. In conclusion, co-expression of IL-15 with IL13RA2-CAR enhances CAR T-cell proliferation and in vivo persistence and achieves durable tumour clearance. IL13RA2-CARIL-15 is being translated into a phase I clinical trial for patients with DMG.

Abstract P367: Brain-Wide Quantification of Amyloid in Transgenic Mice Using Volumetric Imaging and Automated Image Analysis

Localizing and quantifying amyloid in three dimensions furthers understanding of spatial patterns of amyloid deposition and disease progression in experimental models of cerebral amyloid angiopathy (CAA). Serial two-photon tomography (STPT) is ideal for this purpose because it produces 3D datasets of the whole mouse brain in a matter of hours. We present an optimized pipeline for fluorescent whole-brain labeling of amyloid and vasculature, STPT imaging of labeled brains, machine learning-aided classification of vascular and parenchymal amyloid, registration of images to a standard reference atlas, and automated quantification of these signals across brain regions. We used traditional histological sectioning and slide mounted imaging to compare and optimize published methods of fluorescent amyloid and vascular labeling. An intraperitoneal injection of methoxy-X04 delivered the day before euthanasia was chosen to label amyloid. Cerebral vessels are visualized by transcardial perfusion of rhodamine-B isothiocyanate in gelatin. Labeled brains are imaged by STPT and rendered in 3D to appreciate regional amyloid distribution. Volumetric image datasets are quantified using a modification of our custom image analysis pipeline. Pixels are classified as vascular or parenchymal amyloid with supervised machine learning methods. Classified signals are registered into the Allen Institute for Brain Science Common Coordinate Framework version 3.0 for quantification. This strategy produces high resolution volumetric images amenable to automatic quantification of parenchymal and vascular amyloid deposits throughout the brain, appropriate for studies involving large numbers of experimental brains in rodent models of CAA. Figure 1: A 3D rendering of prediction probability maps of a whole labelled mouse brain depicting fluorescent signals that were automatically classified as parenchymal amyloid (cyan), vascular amyloid (yellow) and brain parenchyma.

Is preoperative CA-125 level a predictor of cytoreduction in carcinoma ovary?

Optimal cyto-reduction of advanced EOC has profound impact on the survival and prognosis of patients3. Suboptimal cyto-reduction of advanced ovarian cancer is related to initial tumor bulk which indirectly correlates with serum CA125 levels. Can assessment of level of CA 125 in pre-opererative period predict optimal cyto-reduction is the aim of the study. The mean age of the 70 patients in this study was 49.9 years (SD- 11.75, Range: 21-69 years). Fourty four (63%) had stage III-?V disease. Sixty five (93%) patients had papillary serous ovarian cancer. The serum CA125 levels ranged from 6- 5300 U/ml with median pre-operative serum CA-125 level for all the patients being 259 U/ml (IQR- 682.98U/ml). The level was elevated (>35 U/ml) in 49% of patients with stage ?-? compared with 93% of patients with stage ? or ?V disease. Optimal tumor cyto-reduction was obtained in 61/70 cases (87%) of total patients and in 35/44 (80%) of patients with stage III-?V disease. Diaphragmatic stripping, appendecetomy, recto-sigmoid resection and anastomosis, pelvic peritonectomy, peritoneal stripping/ debulking of peritoneal disease, hemi-colectomy, ileo colic anastomosis and partial nephrectomy. Most of our patients were in intermediate surgical complexity category with a score between 4-7. In 9 patients all belonging to advanced stage, sub-optimal cyto-reduction was done due to multiple co-morbidities, multiple mesenteric lymphnode involvement, multiple small bowel and liver parenchymal deposits and patient not willing for colostomy. An ROC curve analysis showed threshold value of CA125 of 411U/ml. With a cut-off of 411U/ml, 97% of total patients with CA125 levels lower than 411U/ml were able to undergo optimal cyto-reduction. Also, in 76% of patients with CA125 level more than 411U/ml optimal cyto-reduction could be achieved. When only advanced stages (stage III-IV) were considered, with a cut-off of 411U/ml, 88% of patients with CA125 levels lower than 411U/ml were able to undergo op

Using seeding models to understand strain differences from Alzheimer’s disease and pathological aging brains

AbstractBackgroundPathological aging (PA) patients are individuals that are cognitively normal prior to death but have abundant and widespread amyloid‐β (Aβ) deposits. These individuals typically have few cored amyloid deposits with variable levels of diffuse parenchymal deposits and/or vascular deposits. There is little or no inflammatory reaction, neuritic pathology or neurofibrillary tangles in the cortex. Previously, we have shown that total Aβ levels in PA brain lysates were similar to the levels in Alzheimer’s disease (AD) brain lysates, with overlap in the length of the Aβ peptides that were deposited and no major differences in SDS‐stable Aβ oligomeric assemblies. Despite these similarities, it is unclear whether PA represents a prodromal phase of AD or if the amyloid pathology of PA patients represents a less pathogenic variant. To explore this idea, we examined the seeding capacity of AD and PA brain homogenates to induce Aβ deposition and to secondarily induce tau pathology.MethodWe determined the seeding capacity of two AD, four PA and two control brain homogenates in transgenic mice expressing APPswe/ind with and without Tau‐P301L by intracerebral injection at neonatal day 0. At either 6, 9, 12 or 18 months, we assessed Aβ deposition and neurofibrillary pathology in all 4 genotypes by immunohistochemical and biochemical methods. Furthermore we examined the seeding activity of human AD brain lysates in brain slice cultures from APP/Tau transgenic mice.ResultIn our newborn mouse injections, we observed widespread Aβ seeding as evidenced by accelerated onset of diffuse parenchymal Aβ deposition that was largely proportional to the burden of parenchyma deposits in patient brains. In all cases, high levels of pial deposition were observed and Aβ42 was the predominant peptide deposited. Tau pathology was not induced in any animal, and with the absence of neuritic plaques there was no induction of tau positive neurites.ConclusionThis study provides evidence that the Aβ conformers associated with diffuse Aβ deposits in the brains of cognitively normal individuals possess seeding activities similar to those found in the brains of AD patients. The findings are consistent with the idea that pathological aging is on a continuum with AD.

SUN-333 MANAGEMENT OF UROLITHIASIS IN LIVING KIDNEY DONORS

Calcium lithiasis is the most frequently diagnosed renal lithiasis and is associated with a high percentage of patients with metabolic disorders, such as hypercalciuria, hypocitraturia, and hyperoxaluria. Presence of Oxalate-calcium stone in living kidney donor before transplantation may be should make them unselected for kidney donation in some cases We conducted this retrospective study to analyze our experience in management of urolithiasis or hyperoxaluria in living kidney donors. This report is about 3 females aged 49, 43 and 38 years old. All are proposed for kidney donation to their sister. All are not obese, without any medical history and without any pathological symptom such as nephretic colic. We noted no urinary lithiasis on radiographs. We performed a 24-hours urine test, and examined PH, calcium and oxalate. For the first patient, we note a family history of urinary lithiasis and initial nephropathy for the recipient was undetermined. In the 2 other cases, we noted on tomodensitometry renal arterial subcentimeter calculi in 2 cases. In case 3, we noted vitamin D deficiency related hyperparathyroidism. The urine analysis showed in case 1: PH at 5.7, hypercalciuria with Ca/creatinuria at 0.818 mmol/l and weddelite. In patient 2 we noted PH at 5.5, hyperoxaluria at 0.48 mmol/l and oxalate/creatinuria at 0.057. In patient 3 with calculi, we noted PH at 6.6, mild hyperoxaluria with oxalate/creatinuria at 0.035 (normal rate < 0.03). This patient underwent pre transplant extracorporeal shock, hyperdiuresis regimen with stone clearance. She donated a kidney for her sister. After a follow up of 16 months, there was no stone recurrence in donor or recipient. The other 2 patients were refused for kidney donation. This report shows the efficacy of pre transplant extracorporeal shock in living donors with subcentimeter calculi permitting kidney donation after stone clearance. When there is a family history of stone, metabolic disease or donor lumbar pain, screening for hypercalciuria and hyperoxaluria should be performed. There is a controversial if the kidney stone formers can donate a kidney or not. Proper donor selection and follow up is crucial to success. Extracorporeal shock in living donors with subcentimeter calculi can be performed successfully permitting kidney donation after stone clearance. Some stone forming patients are clearly not candidates, such as those with hyperoxaluria, primary hyperoxaluria, or cystinuria or with renal parenchymal deposits.

Shortening heparan sulfate chains prolongs survival and reduces parenchymal plaques in prion disease caused by mobile, ADAM10-cleaved prions.

Cofactors are essential for driving recombinant prion protein into pathogenic conformers. Polyanions promote prion aggregation in vitro, yet the cofactors that modulate prion assembly in vivo remain largely unknown. Here we report that the endogenous glycosaminoglycan, heparan sulfate (HS), impacts prion propagation kinetics and deposition sites in the brain. Exostosin-1 haploinsufficient (Ext1+/-) mice, which produce short HS chains, show a prolonged survival and a redistribution of plaques from the parenchyma to vessels when infected with fibrillar prions, and a modest delay when infected with subfibrillar prions. Notably, the fibrillar, plaque-forming prions are composed of ADAM10-cleaved prion protein lacking a glycosylphosphatidylinositol anchor, indicating that these prions are mobile and assemble extracellularly. By analyzing the prion-bound HS using liquid chromatography-mass spectrometry (LC-MS), we identified the disaccharide signature of HS differentially bound to fibrillar compared to subfibrillar prions, and found approximately 20-fold more HS bound to the fibrils. Finally, LC-MS of prion-bound HS from human patients with familial and sporadic prion disease also showed distinct HS signatures and higher HS levels associated with fibrillar prions. This study provides the first in vivo evidence of an endogenous cofactor that accelerates prion disease progression and enhances parenchymal deposition of ADAM10-cleaved, mobile prions.

Amyloid and Tau in Neurodegeneration

Background and Hypothesis:Familial British dementia (FBD) and familial Danish dementia (FDD) are two autosomal dominant neurodegenerative diseases caused by mutations in the BRI2 gene. FBD and FDD are characterized by widespread cerebral amyloid angiopathy (CAA), parenchymal amyloid deposition, and neurofibrillary tangles.Previous studies have shown that accumulation of Danish amyloid peptide (ADan) induces hyperphosphorylation of tau and cell cytotoxicity; however, progress in our understanding of the interaction between ADan and tau has been hindered by the lack of antibodies against ADan. In addition, whether the British amyloid peptide (ABri) has a similar effect on tau phosphorylation remains unknown. The main goals of our project are to generate monoclonal antibodies against ADan and to test whether the ABri peptide induces hyperphosphorylation of tau and cell cytotoxicity.&#x0D; Experimental Design or Project Methods:A peptide homologous to the C-terminus of the ADan peptide was used to immunize 5 mice. Serum samples were tested from mice with high ELISA titers and the best 2 animals were used for cell fusion. Cell culture supernatants were screened by ELISA, western blot and immunohistochemistry. ABri peptides were used as negative controls.HEK cells expressing human tau were used to assess the interaction between ABri and tau. We performed immunocytochemistry and Western blot analysis to investigate tau phosphorylation.&#x0D; Results:A total of 11 clones tested positive by western blot. 3 clones tested positive for ABri and were discarded. 5 clones were tested by immunohistochemistry. 3 of the positive clones will be used for subcloning to complete clonality. Work in progress on the interaction between ABri and tau will determine whether ABri induces tau hyperphosphorylation in cell culture.&#x0D; Conclusion and Potential Impact:Our work will provide novel tools to study the interaction between amyloid and tau in neurodegenerative diseases and may uncover possible new targets for pharmaceutical intervention in tauopathies.

Brain ApoA-I, ApoJ and ApoE Immunodetection in Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA) is a common cause of lobar intracerebral hemorrhage (ICH) in elderly individuals and it is the result of the cerebrovascular deposition of beta-amyloid (Aβ) protein. CAA is frequently found in patients with Alzheimer's disease (AD), although it has an independent contribution to the cognitive deterioration associated with age. Specific apolipoproteins (Apo) have been associated with Aβ fibrillization and clearance from the brain. In this regard, in the present study, we analyzed the brain levels of ApoE, ApoA-I, and ApoJ/clusterin in autopsy brains from 20 post-mortem cases with CAA type I, CAA type II, with parenchymal Aβ deposits or without Aβ deposits. Our objective was to find a possible differential pattern of apolipoproteins distribution in the brain depending on the CAA pathological presentation. The protein expression levels were adjusted by the APOE genotype of the patients included in the study. We found that ApoE and ApoJ were abundantly present in meningeal, cortical, and capillary vessels of the brains with vascular Aβ accumulation. ApoE and ApoJ also deposited extracellularly in the parenchyma, especially in cases presenting Aβ diffuse and neuritic parenchymal deposits. In contrast, ApoA-I staining was only relevant in capillary walls in CAA type I cases. On the other hand, ICH was the principal cause of death among CAA patients in our cohort. We found that CAA patients with ICH more commonly had APOEε2 compared with CAA patients without ICH. In addition, patients who suffered an ICH presented higher vascular ApoE levels in brain. However, higher ApoE presence in cortical arteries was the only independent predictor of suffering an ICH in our cohort after adjusting by age and APOE genotype. In conclusion, while ApoE and ApoJ appear to be involved in both vascular and parenchymal Aβ pathology, ApoA-I seems to be mainly associated with CAA, especially in CAA type I pathology. We consider that our study helps to molecularly characterize the distribution subtypes of Aβ deposition within the brain.

A distinct brain beta amyloid signature in cerebral amyloid angiopathy compared to Alzheimer’s disease

Cerebral amyloid angiopathy (CAA) is a type of vascular disease present in more than 50% of demented elderly and more than 80% of Alzheimer’s disease (AD) patients. Both CAA and AD are characterized by extracellular Aβ deposits with the distinction that CAA has vascular deposits while AD has amyloid plaques. In this study, we used immunoprecipitation (IP) in combination with mass spectrometry (MS) to test the hypothesis that the Aβ peptide pattern differs between subjects having Aβ plaque pathology only and subjects with Aβ plaque pathology together with CAA pathology. Occipital lobes from 12 AD brains, ranging from no CAA to severe CAA, were extracted using 70% formic acid followed by IP-MS analysis. The Aβ peptide pattern differed greatly between subjects with no CAA compared to subjects with CAA. In cases with CAA, the most abundant Aβ peptides ended at amino acid 40 including Aβ1-40 (P = .048) and Aβ 2–40 (P = .0253) which were significantly increased compared to cases with no CAA. This was in contrast to subjects with no CAA where the most abundant Aβ peptides ended at amino acid 42 of which Aβ1-42 (P = .0101) and Aβ2-42 (P = .0051) as well as the pyroglutamate (pGlu)-modified peptides pGlu Aβ3-42 (P = .0177), and pGlu Aβ11-42 (P = .0088) were significantly increased compared to CAA subjects. The results are in line with earlier immunohistochemistry data and show that the molecular composition of the Aβ deposits found in blood vessels are different to the parenchymal deposits, suggesting they arise from distinct pathogenic pathways. This information may be useful in the development of pathology-specific biomarkers.

Modified amyloid variants in pathological subgroups of β-amyloidosis.

ObjectiveAmyloid β (Aβ) depositions in plaques and cerebral amyloid angiopathy (CAA) represent common features of Alzheimer's disease (AD). Sequential deposition of post‐translationally modified Aβ in plaques characterizes distinct biochemical stages of Aβ maturation. However, the molecular composition of vascular Aβ deposits in CAA and its relation to plaques remain enigmatic.MethodsVascular and parenchymal deposits were immunohistochemically analyzed for pyroglutaminated and phosphorylated Aβ in the medial temporal and occipital lobe of 24 controls, 27 pathologically‐defined preclinical AD, and 20 symptomatic AD cases.ResultsSequential deposition of Aβ in CAA resembled Aβ maturation in plaques and enabled the distinction of three biochemical stages of CAA. B‐CAA stage 1 was characterized by deposition of Aβ in the absence of pyroglutaminated Aβ N3pE and phosphorylated Aβ pS8. B‐CAA stage 2 showed additional Aβ N3pE and B‐CAA stage 3 additional Aβ pS8. Based on the Aβ maturation staging in CAA and plaques, three case groups for Aβ pathology could be distinguished: group 1 with advanced Aβ maturation in CAA; group 2 with equal Aβ maturation in CAA and plaques; group 3 with advanced Aβ maturation in plaques. All symptomatic AD cases presented with end‐stage plaque maturation, whereas CAA could exhibit immature Aβ deposits. Notably, Aβ pathology group 1 was associated with arterial hypertension, and group 2 with the development of dementia.InterpretationBalance of Aβ maturation in CAA and plaques defines distinct pathological subgroups of β‐amyloidosis. The association of CAA‐related Aβ maturation with cognitive decline, the individual contribution of CAA and plaque pathology to the development of dementia within the defined Aβ pathology subgroups, and the subgroup‐related association with arterial hypertension should be considered for differential diagnosis and therapeutic intervention.

Differential Effect of APOE Alleles on Brain Glucose Metabolism in Targeted Replacement Mice: An [18F]FDG-μPET Study

Background:The Apolipoprotein E (ApoE) alleles ɛ2, ɛ3, and ɛ4 are known to differentially modulate cerebral glucose metabolism and the risk for Alzheimer’s disease (AD) via both amyloid-β (Aβ)-dependent and independent mechanisms.Objective:We investigated the influence of ApoE on cerebral glucose metabolism in humanized APOE Targeted Replacement (TR) mice at ages that precede the comparison of Aβ parenchymal deposits in APOE4-TR mice.Methods:Fludeoxyglucose ([18F]FDG) positron emission tomography (PET) measures were performed longitudinally in homozygous APOE-TR mice (APOE2, APOE3, APOE4; n = 10 for each group) at 3, 5, 11, and 15 months. Results were quantified using standard uptake values and analyzed statistically using a linear mixed effects model. Levels of the Aβ40 and Aβ42 peptides were quantified ex vivo using enzyme-linked immunosorbent assay (ELISA) at 15 months in the same animals.Results:APOE2 mice (versus APOE3) showed a significant increase in glucose metabolism starting at 6 months, peaking at 9 months. No evidence of hypometabolism was apparent in any region or time point for APOE4 mice, which instead displayed a hypermetabolism at 15 months. Whole brain soluble Aβ40 and Aβ42 levels were not significantly different between genotypes at 15 months.Conclusions:Introduction of human APOE alleles ɛ2 and ɛ4 is sufficient to produce alterations in brain glucose metabolism in comparison to the control allele ɛ3, without a concomitant alteration in Aβ40 and Aβ42 levels. These results suggest novel Aβ-independent metabolic phenotypes conferred by ɛ2 and ɛ4 alleles and have important implications for preclinical studies using TR-mice.

Abstract WMP82: Exosomes Derived From Cerebral Endothelial Cells of Young Adult Rats Reduced Cognitive Deficits in Aged Diabetic Rats

Introduction: Diabetes mellitus (DM) is associated with cognitive decline and dementia in the elderly. The glymphatic system mediates clearance of the interstitial solutes in the brain by exchange of cerebrospinal and interstitial fluid (CSF and ISF). We recently demonstrated that DM in aged rat induces impairment of the glymphatic system and cognitive decline. Exosomes, membrane vesicles, mediate intercellular communication by transferring their cargo into recipient cells. The present study investigated whether cerebral endothelial exosomes (CEE) ameliorate glymphatic system impairment and improve cognitive function in aged DM rats. Methods and Results: DM was induced in male Wistar rats (13 months, n=48) by injection of nicotinamide and streptozotocin. Two months after DM, rats were treated with CEE (1x10 11 exosomes/rat, IV) twice a week for 4 weeks. Age matched DM and non-DM rats were used as controls. CEE were harvested from the cultured cerebral endothelial cells of health young adult rats. Exchanges of CSF and ISF were measured by intracisternal injection of fluorescent tracer, Texas Red-dextran (TR, 3kD). Confocal microscopic analysis of brain slices revealed a progressive slowdown of ISF clearance in the hippocampi, assessed by retention of TR starting at 2.5 fold at 2M (13±5 vs 5±3% of area) and increasing to 4 fold at 4M (21±4 vs 5±2%) of DM. Paravascular amyloid β (Aβ) accumulation was only detected at 4M of DM. The CEE treatment significantly (p&lt;0.05) reduced TR retention (10±4%) at 4M of DM and also decreased Aβ accumulation (2±1 vs 6±2/mm 2 ) and parenchymal fibrin deposition (9±5 vs 23±5/mm 2 ) compared to untreated DM rats. Moreover, the CEE treatment significantly improved hippocampal related learning and memory measured by the Morris Water Maze and odor-based novelty recognition for olfactory memory, without altering the glucose level. In vitro, cerebral endothelial cells isolated from 2M DM rats exhibited substantial dysfunction as measured by capillary-like tube formation and cell migration, whereas incubation with the CEE substantially reversed endothelial dysfunction. Conclusions: The CEE treatment reduces DM-induced glymphatic and cerebral endothelial dysfunctions, leading to improvement of cognitive function in aged DM rats.

Molecular interaction between type 2 diabetes and Alzheimer's disease through cross-seeding of protein misfolding.

Numerous epidemiological studies have shown a significantly higher risk for development of Alzheimer’s disease (AD) in patients affected by type 2 diabetes (T2D), but the molecular mechanism responsible for this association is presently unknown. Both diseases are considered protein misfolding disorders associated to the accumulation of protein aggregates; amyloid-beta (Aβ) and tau in the brain during AD, and islet amyloid polypeptide (IAPP) in pancreatic islets in T2D. Formation and accumulation of these proteins follows a seeding-nucleation model, where a misfolded aggregate or “seed” promotes the rapid misfolding and aggregation of the native protein. Our underlying hypothesis is that misfolded IAPP produced in T2D potentiates AD pathology by cross-seeding Aβ, providing a molecular explanation for the link between these diseases. Here, we examined how misfolded IAPP affects Aβ aggregation and AD pathology in vitro and in vivo. We observed that addition of IAPP seeds accelerates Aβ aggregation in vitro in a seeding-like manner and the resulting fibrils are composed of both peptides. Transgenic animals expressing both human proteins exhibited exacerbated AD-like pathology compared to AD transgenic mice or AD transgenic animals with type-1 diabetes (T1D). Remarkably, IAPP colocalized with amyloid plaques in brain parenchymal deposits, suggesting these peptides may directly interact and aggravate the disease. Furthermore, inoculation of pancreatic IAPP aggregates into the brains of AD transgenic mice resulted in more severe AD pathology and significantly greater memory impairments than untreated animals. This data provides a proof-of-concept for a new disease mechanism involving the interaction of misfolded proteins through cross-seeding events which may contribute to accelerate or exacerbate disease pathogenesis. Our findings could shed light on understanding the linkage between T2D and AD, two of the most prevalent protein misfolding disorders.

Diagnosis and pathology of colibacillosis in Asian brown tortoise (Manouria emys phayrei)

Mortality in two female tortoise of Zoological Garden Aizawl was investigated. On the basis of morpho-anatomy it was found that the tortoise belonged to species Manouria emys phayrei which is an endangered species. Thorough necropsy examination revealed oophoritis and egg peritonitis. Bacterial isolation from ova yielded pure colonies of Escherichia coli which was serotyped as O2. Antibiotic sensitivity test revealed that it is susceptible to most of the commonly used drugs including Enrofloxacin, Azotreonam, Ceftriazone, Streptomycin, Ceftazidime, Cefalexin, Cefixime, Cefotaxime and Piperacellin. Histopathological examination revealed microthrombosis in liver sinusoids and hyaline proteinaceous deposits in parenchyma of spleen. The isolates could not be classified under pathotypes by detection of virulence genes by Polymerase chain reaction. The findings showed that E. coli could cause egg peritonitis and colisepticemia like lesions in tortoise. This is the first report of E. coli associated collibacillosis in Asian brown tortoise.

Abstract 1803: A truncated Plasminogen Activator Inhibitor-1 protein protects from pulmonary fibrosis mediated by irradiation in a murine model

Abstract Background: Fibrosis can develop as a late side effect of radiation exposure in a variety of tissues, including lung and skin. Late radiation injury and fibrosis are characterized by parenchymal cell depletion, inflammation, senescence, fibroblast proliferation, and excessive deposition of collagen. Plasminogen activator inhibitor 1 (PAI-1) is a critical mediator of cellular senescence and fibrin stabilization, and its expression is increased in experimental fibrosis models. We sought to determine if inhibition of PAI-1 signaling with a recombinant truncated protein (rPAI-123) would protect from the development of radiation-induced lung injury. Methods: C57Bl/6 mice received intraperitoneal injections of rPAI-123 (5.4 μg/kg/day) or vehicle (PBS) for 18 weeks beginning two days prior to radiation exposure. Cohorts of mice treated with rPAI-123 or vehicle were exposed to thoracic irradiation in 5 daily fractions of 6 Gy (RT), and followed for survival (n = 8 per group) and tissue collection (n = 3 per each time point). Histologic changes in irradiated lungs were evaluated by Masson-Trichrome staining at 19 weeks after RT. Senescence was assessed with staining for beta-galactosidase activity in lung tissue and primary pneumocytes. To define the roles of rPAI-123 in the fibroproliferative response, cell proliferation (MTT assay) and collagen deposition (Hydroxyproline assay) were examined in a mouse fibroblast cell line in vitro. Results: Administration of rPAI-123 increased C57Bl/6 mice survival from 37.5% to 62.5% at 19 weeks after radiation exposure. At 19 weeks after irradiation, hydroxyproline content was markedly decreased in mice received rPAI-123 compared to mice received vehicle (RT+rPAI-123: 56.2±10.79, RT+vehicle: 84.97±2.98, μg/lung respectively, p = 0.001 between RT+vehicle and RT+rPAI-123). C57Bl/6 mice exposed to RT+vehicle had dense foci of subplueral fibrosis at 19 weeks, whereas the lungs of mice exposed to RT+rPAI-123 were largely devoid of fibrotic foci. Cellular senescence in response to radiation was significantly decreased by rPAI-123 treatment in primary type2 pneumocyte culture (2-fold reduction at 5 days after RT, p = 0.036), and in lung tissues (&amp;gt;2-fold reduction at 4, 8, and 16 weeks after RT, p&amp;lt;0.001 at each time point). Treatment of NIH-3T3 fibroblasts with rPAI-123 resulted in decreased collagen production, but had no effect on proliferation.Conclusions: These studies identify that rPAI-123 has a novel protection mechanism against radiation-induced fibrosis in murine lungs due to its ability to reduce senescence in type2 pneumocytes, and the potential to be an effective therapy option for radiation induced fibrosis. Citation Format: Eunjoo Chung, Ayla White, Bradley T. Scroggins, Grace B. McKay-Corkum, Mary Jo Mulligan-Kehoe, Deborah E. Citrin. A truncated Plasminogen Activator Inhibitor-1 protein protects from pulmonary fibrosis mediated by irradiation in a murine model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1803. doi:10.1158/1538-7445.AM2015-1803

Benign Lymphoepithelial Lesion of Parotid Gland and Secondary Amyloidosis as Concurrent Manifestations in Sjögren's Syndrome.

Benign Lymphoepithelial Lesion of Parotid Gland and Secondary Amyloidosis as Concurrent Manifestations in Sjögren's Syndrome.

Abstract W MP52: Treatment With N-acetyl-seryl-aspartyl-lysyl-proline Improves Neurological Functional Recovery in Diabetic Middle Aged Rats After Embolic Stroke

Background and Purpose: Diabetes mellitus (DM) is a common metabolic disease among middle-aged and older populations, which leads to an increase of stroke incidence and poor stroke recovery. The present study investigated the effect of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) on stroke recovery in middle aged DM rats after embolic stroke. Methods: Male Wistar rats at age of 13 months were subjected to nicotinamide (NTM) and streptozotocin (STZ) injection . Rats with confirmed hyperglycemia status were subjected to embolic middle cerebral artery occlusion (MCAO) 30d after STZ-NTM injection. AcSDKP (2.4mg/kg) or saline were subcutaneously infused for 3d starting at 1d after stroke (n=12/group). Results: AcSDKP treatment significantly improved neurological functional recovery measured by adhesive removal test, foot-fault test, and modified neurological severity score starting at 21d after MCAO compared with saline treated rats. In addition, AcSDKP substantially reduced spatial learning deficits measured by the Morris water maze (41±4 vs 33±3% of time spent in the correct quadrant) 35d after MCAO. AcSDKP treatment significantly reduced vascular and parenchymal fibrin deposition (43±9 vs 92±11/mm 2 ) in the ipsilateral hippocampus and peri-infarct corpus callosum (CC), which was associated with reduction of IL-1 receptor associated kinase-1(IRAK1) immunoreactive vessels (95±13 vs 47±11/mm 2 ) 7d after MCAO (n=4/group). AcSDKP treatment did not reduce infarct volume, but robustly increased myelinated axonal density (14±4 vs 8±3% in hippocampus, 36±6 vs 27±7% in CC) and oligodendrocyte density (71±18 vs 46±16/mm 2 in hippocampus, 526±113 vs 394±107/mm 2 in CC) 35d after MCAO (n=8/group). In vitro, high glucose increased protein level of IRAK1 and NF-κB in primary cerebral endothelial cells, whereas AcSDKP suppressed high glucose-increased IRAK1 and NF-κB protein levels by 50%. Conclusions: AcSDKP promotes ischemic brain repair by reducing neurovascular damage in the hippocampus and white matter, which likely contributes improvement of neurological functional recovery in middle-aged DM rats. IRAK1/NF-κB signals suppressed by AcSDKP may be involved these processes.