Retromer Proteins Reduced in Down Syndrome and the Dp16 Model: Impact of APP Dose and Preclinical Studies of a γ-Secretase Modulator.

OBJECTIVE: The retromer complex plays an essential role in intracellular endosomal sorting. Deficits in the retromer complex are linked to enhanced Aß production. The levels of the components of the retromer complex are reported to be downregulated in Alzheimer disease (AD). Down syndrome (DS) shares neuropathological features with AD. Recent evidence points to dysregulation of the retromer complex in DS. The mechanisms underlying retromer deficits in DS and AD are poorly understood. METHODS: We measured the levels of retromer components in the frontal cortex of cases of DS-AD (AD in DS) as well as DS; the frontal cortex of a person partially trisomic (PT-DS) for human chromosome 21 (HSA21), whose genome had only the normal 2 copies of the APP gene, was also examined. We also analyzed these proteins in the Dp16 mouse model of DS. To further explore the molecular mechanism for changes in the retromer complex, we treated Dp16 mice with a γ-secretase modulator (GSM; 776890), a treatment that reduces the levels of Aß42 and Aß40. RESULTS: We found VPS26A, VPS26B, and VPS29, but not VPS35, were significantly reduced in both DS and DS-AD, but not in PT-DS. Downregulation of VPS26A, VPS26B, and VPS29 was recapitulated in the brains of old Dp16 mice (at 16 months of age) and required increased App gene dose. Significantly, GSM treatment completely prevented reductions of the retromer complex. INTERPRETATION: Our studies point to increased APP gene dose as a compromising retromer function in DS and suggest a causal role for Aß42 and Aß40. ANN NEUROL 2023;94:245-258.

Usefulness and Limitations of Cryopreservation for Immunocytochemical Staining of Canine Cytological Specimens for Detection of Cytokeratin and Vimentin.

Immunocytochemistry is an advanced diagnostic tool for identifying the origin of tumor cells. This study aimed to highlight the usefulness of cryopreserved, air-dried cytological samples in detecting cytokeratin and vimentin. Air-dried cytological smear samples were prepared from a total of 39 resected canine tumors and stored in a medical freezer without fixation. The duration of cryopreservation ranged from 2 to 56 months. The same tumors were processed for routine histopathological examination. Based on the morphological diagnosis, cryopreserved FNA smears from epithelial tumors were stained by enzymatic immunocytochemistry (ICC) for cytokeratin; those from mesenchymal and melanocytic tumors were stained by ICC for vimentin. To ascertain the positivity of tumor cells to the selected markers, tissue paraffin-embedded sections were also stained by immunohistochemistry (IHC) for the same markers. Immunoreactivity for cytokeratin was detected in cryopreserved cytological smears for a maximum of 46 months. Immunoreactivity for vimentin was clearly detected for 33 months. Smears stored at room temperature for 1 week did not show any signals under immunocytochemical examination. Thus, immunocytochemistry for cytokeratin and vimentin can be safely applied to air-dried smears cryopreserved in a freezer for at least 33 months.

Impact of increased APP gene dose in Down syndrome and the Dp16 mouse model.

INTRODUCTION: People with Down syndrome (DS) are predisposed to Alzheimer's disease (AD). The amyloid hypothesis informs studies of AD. In AD-DS, but not sporadic AD, increased APP copy number is necessary, defining the APP gene dose hypothesis. Which amyloid precursor protein (APP) products contribute needs to be determined. METHODS: Brain levels of full-length protein (fl-hAPP), C-terminal fragments (hCTFs), and amyloid beta (Aß) peptides were measured in DS, AD-DS, non-demented controls (ND), and sporadic AD cases. The APP gene-dose hypothesis was evaluated in the Dp16 model. RESULTS: DS and AD-DS differed from ND and AD for all APP products. In AD-DS, Aß42 and Aß40 levels exceeded AD. APP products were increased in the Dp16 model; increased APP gene dose was necessary for loss of vulnerable neurons, tau pathology, and activation of astrocytes and microglia. DISCUSSION: Increases in APP products other than Aß distinguished AD-DS from AD. Deciphering AD-DS pathogenesis necessitates deciphering which APP products contribute and how.

Immunocytochemical evaluation of epithelial-mesenchymal transition in epithelial tumors of dogs and cats.

Epithelial-mesenchymal transition (EMT) plays a crucial role in metastasis of epithelial tumors; however, it is challenging to detect EMT by cytology. In the present study, EMT was visualized by fluorescence-immunocytochemistry (FICC). Air-dried smears from epithelial tumors of dogs (n=22) and cats (n=9) were stained using mouse monoclonal anti-E-cadherin and rabbit monoclonal anti-vimentin antibodies. Enzymatic immunohistochemistry (IHC) revealed that 51.6% (8/22 in dogs, 8/9 in cats) of the cases showed EMT. In dogs, FICC could detect EMT in 62.5% (5/8) of those cases. In cats, FICC could detect EMT in 100% (8/8) of the cases. In conclusion, the present FICC method could successfully detect EMT using conventional air-dried cytology smear slides.

A rapid multiple immunofluorescence method for the simultaneous detection of CD20 and CD3 in canine and feline cytological samples.

CD20 and CD3 are considered reliable markers for B and T cells, respectively. This study aimed to develop a rapid multiple immunofluorescence (RMIF) method for the detection of CD20 and CD3 on a single cytology slide. Air-dried smears were prepared using samples collected from dogs (n=26) and cats (n=6). Immunosignal detection using the newly developed method required 60 min. Clear immunosignals for CD20 and CD3 were detected in 24 of 26 samples in dogs and in all 6 cats. As the RMIF (CD20/CD3) method can detect markers of both B and T cells simultaneously on a single cytology smear, it would be an efficient tool for the immunophenotyping of canine and feline lymphoma samples.

Preclinical validation of a potent γ-secretase modulator for Alzheimer's disease prevention.

A potent γ-secretase modulator (GSM) has been developed to circumvent problems associated with γ-secretase inhibitors (GSIs) and to potentially enable use in primary prevention of early-onset familial Alzheimer's disease (EOFAD). Unlike GSIs, GSMs do not inhibit γ-secretase activity but rather allosterically modulate γ-secretase, reducing the net production of Aß42 and to a lesser extent Aß40, while concomitantly augmenting production of Aß38 and Aß37. This GSM demonstrated robust time- and dose-dependent efficacy in acute, subchronic, and chronic studies across multiple species, including primary and secondary prevention studies in a transgenic mouse model. The GSM displayed a >40-fold safety margin in rats based on a comparison of the systemic exposure (AUC) at the no observed adverse effect level (NOAEL) to the 50% effective AUC or AUCeffective, the systemic exposure required for reducing levels of Aß42 in rat brain by 50%.

Argyrophilic nucleolar organizer regions staining for cytology smears in dogs and cats.

The argyrophilic nucleolar organizer regions (AgNORs) are cellular proliferation markers, crucial for predicting the clinical course and aggressiveness of tumors. The purpose of this study was to establish an easy and practical AgNOR staining method in the cytology of dogs and cats. Air-dried cytological slides were prepared from dogs (n=14) and cats (n=12). Acetone, formalin, ethanol and methanol were tested as fixatives for AgNOR staining. Subsequently, various methods of Romanowsky-based counterstains were tested before and after AgNOR staining. Clear and strong AgNOR spots were observed with all fixatives, and post-May-Grünwald staining was the best counterstaining method. The established method showed clear AgNOR spots even in the long-term storage samples and Romanowsky-stained ones.

Down syndrome.

Down syndrome (DS; Trisomy 21) is the most common chromosomal disorder in humans. It has numerous associated neurologic phenotypes including intellectual disability, sleep apnea, seizures, behavioral problems, and dementia. With improved access to medical care, people with DS are living longer than ever before. As more individuals with DS reach old age, the necessity for further life span research is essential and cannot be overstated. There is currently a scarcity of information on common medical conditions encountered as individuals with DS progress into adulthood and old age. Conflicting information and uncertainty about the relative risk of dementia for adults with DS is a source of distress for the DS community that creates a major obstacle to proper evaluation and treatment. In this chapter, we discuss the salient neurologic phenotypes of DS, including Alzheimer's disease (AD), and current understanding of their biologic bases and management.

Multi-level Modulation of Light Signaling by GIGANTEA Regulates Both the Output and Pace of the Circadian Clock.

Integration of environmental signals with endogenous biological processes is essential for organisms to thrive in their natural environment. Being entrained by periodic environmental changes, the circadian clock incorporates external information to coordinate physiological processes, phasing them to the optimal time of the day and year. Here, we present a pivotal role for the clock component GIGANTEA (GI) as a genome-wide regulator of transcriptional networks mediating growth and adaptive processes in plants. We provide mechanistic details on how GI integrates endogenous timing with light signaling pathways through the global modulation of PHYTOCHROME-INTERACTING FACTORs (PIFs). Gating of the activity of these transcriptional regulators by GI directly affects a wide array of output rhythms, including photoperiodic growth. Furthermore, we uncover a role for PIFs in mediating light input to the circadian oscillator and show how their regulation by GI is required to set the pace of the clock in response to light-dark cycles.

Rapid multiple immunofluorescent staining for the simultaneous detection of cytokeratin and vimentin in the cytology of canine tumors.

BACKGROUND: Immunocytochemistry (ICC) is utilized as an advanced technique in veterinary cytology. In tumor diagnosis, cytokeratin and vimentin are markers used to distinguish the origin of tumor cells. Standard enzyme-based ICC has limitations in clinical use; and therefore, more convenient and reliable methods are needed. OBJECTIVES: The purpose of this study was to develop a rapid multiple immunofluorescent (RMIF) detection method for dual cytokeratin and vimentin staining on cytology slides in dogs. METHODS: Air-dried smear samples from solid tumors and sediments of pleural effusions were prepared from dogs (n = 14) that were admitted to the Veterinary Teaching Hospital, Kagoshima University, Japan. Mouse monoclonal anti-human cytokeratin (AE1/AE3) and rabbit monoclonal anti-human vimentin (SP20) antibodies were used as primary antibodies, followed by staining with Alexa Fluor-conjugated secondary antibodies. Staining using the RMIF method was compared with enzyme-based ICC staining. RESULTS: Rapid multiple immunofluorescent immunostaining was clear and specific in the evaluated smears, whereas the enzyme-based ICC showed nonspecific signals. By using the RMIF staining method, epithelial cells, mesenchymal cells, and mesothelial cells could be classified on a single smear of a pleural effusion. In smears of lymph nodes with epithelial tumor metastases, the RMIF method successfully detected metastatic epithelial tumor cells. CONCLUSIONS: The RMIF method might be a useful tool for diagnostic cytology in veterinary medicine.

Dysregulation of neurotrophin signaling in the pathogenesis of Alzheimer disease and of Alzheimer disease in Down syndrome.

Neurotrophic factors, including the members of the neurotrophin family, play important roles in the development and maintenance of the nervous system. Trophic factor signals must be transmitted over long distances from axons and dendrites to the cell bodies of neurons. A mode of signaling well suited to the challenge of robust long distance signaling is the signaling endosome. We review the biology of signaling endosomes and the "signaling endosome hypothesis". Evidence for disruption of signaling endosome function in disorders of the nervous system is also reviewed. Changes in endosome structure in Alzheimer disease (AD) and Down syndrome (DS) are present early in these disorders. Data for the APP products responsible are reviewed and the consequent changes in signaling from endosomes discussed. We conclude by pointing to the need for additional studies to explore the biology of signaling endosomes in normal neurons and to elucidate their role in the pathogenesis of neurodegeneration.

Paraffin immunofluorescence for detection of immune complexes in renal biopsies: an efficient salvage technique for diagnosis of glomerulonephritis in dogs.

BACKGROUND: Renal biopsy is an essential tool for the diagnosis of proteinuric kidney diseases in dogs, and evaluation of immune complexes (IC) by immunofluorescence (IF) of frozen sections (IF-F) is required for the diagnosis of IC-mediated glomerulonephritis (ICGN). However, the use of frozen sections from renal biopsies can have limitations. The aim of this study was to develop a reliable IF method using formalin-fixed and paraffin-embedded (FFPE) sections to detect ICs in dog ICGN. METHODS: Renal biopsy specimens were obtained from dogs with protein-losing nephropathies. FFPE sections were prepared, and eight antigen retrieval pretreatment protocols were performed: digestion with trypsin, microwave (MW) heating in citrate buffer (MW-CB; pH 6.0), MW heating in Tris-EDTA buffer (MW-TEB; pH 9.0), as well as combinations of the above, and a non-treated control. RESULTS: A combination of trypsin for 30 min (Try-30) and MW-TEB; pH 9.0 was the most effective antigen retrieval pretreatment, with clear positive signals for IgG, IgA, IgM, and C3 detected by IF-FFPE. Granular signals, an important diagnostic indicator of ICGN, were clearly observed by both IF-F and IF-FFPE after combined pretreatment with Try-30 and MW-TEB, and IgG, IgA, IgM, and C3 signals were almost completely matched in all samples by IF-F and IF-FFPE. CONCLUSION: IF-FFPE with Try-30 and MW-TEB pretreatment is a valuable technique for the diagnosis of renal diseases in dogs. This method could be an efficient tool when standard IF-F cannot be used, or does not provide useful results due to lack of glomeruli in the specimens for IF-F.

Acquired Fanconi syndrome in two dogs following long-term consumption of pet jerky treats in Japan: case report.

Renal Fanconi syndrome has recently been associated with the ingestion of pet jerky treats from China in mostly small breed dogs in North America, Australia and Europe. We report here about two dogs with Fanconi syndrome following pet jerky treats exposure in Japan. A mixed-breed dog and a French bulldog showed weight loss, polyuria and polydipsia. For years, the owners had been feeding large quantities of pet jerky treats containing chicken prepared in China. Diagnostics revealed glycosuria without hyperglycemia, severe aminoaciduria, and in one case also ketonuria, hypokalemia and metabolic acidosis. A diagnosis of Fanconi syndrome associated with long-term consumption of Chinese pet jerky treats was made. Both dogs recovered fully following withdrawal of the pet jerky treats and supportive care. Fanconi syndrome of dogs in association with the consumption of pet jerky treats of Chinese origin can cause a broad proximal tubular defect with glycosuria and generalized amino aciduria, and should be also considered in Asia. Jerky treats associated Fanconi syndrome can be completely reversible following withdrawal of the treats and supportive care to correct the metabolic abnormalities.

Rapid immunocytochemistry for the detection of cytokeratin and vimentin: assessment of its diagnostic value in neoplastic diseases of dogs.

BACKGROUND: Immunocytochemistry (ICC) is an advanced diagnostic technique used in the field of veterinary cytology. We recently developed a rapid ICC method for the detection of cytokeratin and vimentin in dogs, which helps to determine whether tumor cells are of epithelial or nonepithelial origin. However, the diagnostic value of this rapid ICC method in neoplastic diseases of dogs has not been assessed yet. OBJECTIVES: The aim of the present study was to assess the diagnostic accuracy of rapid ICC compared to standard immunohistochemistry (IHC). METHODS: Air-dried smear samples and formalin-fixed paraffin sections were prepared from tumors excised from dogs (n = 30). Immunosignals for cytokeratin and vimentin were detected in smear samples by rapid ICC, and in paraffin sections by standard IHC. Signals in smear samples detected by rapid ICC were compared with positive staining in paraffin sections detected by standard IHC and analyzed for statistical significance (kappa statistic). RESULTS: Rapid ICC detected specific immunosignals in 25/30 cases (83.3%), and nonspecific signals were detected in 5/30 cases. Statistical analysis revealed fair agreement in epithelial tumors (n = 16) with cytokeratin (κ = 0.236) and vimentin (κ = 0.294). In nonepithelial tumors (n = 14), almost perfect agreement was demonstrated with cytokeratin (κ = 0.857) and vimentin (κ = 0.857). CONCLUSIONS: The rapid ICC method can be a useful tool for the diagnostic cytology of neoplastic tissues in dogs.

Degenerative myelopathy in the Collie breed: a retrospective immunohistochemical analysis of superoxide dismutase 1 in an affected Rough Collie, and a molecular epidemiological survey of the SOD1: c.118G>A mutation in Japan.

Canine degenerative myelopathy (DM) is an adult-onset, progressive neurodegenerative disease that occurs in multiple dog breeds. A DM-associated mutation of the canine superoxide dismutase 1 (SOD1) gene, designated as c.118G>A (p.E40K), has been implicated as one of pathogenetic determinants of the disease in many breeds, but it remains to be determined whether the c.118G>A mutation is responsible for development or progression of DM in Collies. Previously, a Rough Collie was diagnosed clinically and histopathologically as having DM in Japan, suggesting the possibility that the Collie breed may be predisposed to DM due to the high frequency of c.118G>A in Japan. In this study, accumulation and aggregate formation of SOD1 protein were retrospectively demonstrated in the spinal cord of the DM-affected dog by immunohistochemical analysis. Furthermore, a molecular epidemiological survey revealed a high carrier rate (27.6%) and mutant allele frequency (0.138) of c.118G>A in a population of Collies in Japan, suggesting that the Collie breed may be predisposed to DM associated with c.118G>A, and the prevention of DM in Collies in Japan should be addressed through epidemiological and genetic testing strategies.

Low expression of cyclooxygenase-2 in chronic kidney disease in young dogs.

Chronic kidney disease (CKD) often results in end-stage renal failure in young dogs; however, the pathogenesis of this disease is not established. This study investigated renal expression of cyclooxygenase (COX)-1 and COX-2 proteins in three dogs with chronic kidney disease by immunohistochemistry. Histopathology showed asynchronous differentiation of renal tissues, including immature glomeruli. COX-1 signals were not detected in diseased or normal kidneys. COX-2 signals were low or undetectable in diseased kidneys, while normal kidneys showed clear positive signals in the macula densa (MD). Quantitative scores of COX-2 in diseased kidneys were significantly lower than those in normal kidneys. These findings demonstrate low renal COX-2 expression in CKD in young dogs, but whether this is correlated with disease pathogenesis remains unclear.

Amyloid precursor protein-mediated endocytic pathway disruption induces axonal dysfunction and neurodegeneration.

The endosome/lysosome pathway is disrupted early in the course of both Alzheimer's disease (AD) and Down syndrome (DS); however, it is not clear how dysfunction in this pathway influences the development of these diseases. Herein, we explored the cellular and molecular mechanisms by which endosomal dysfunction contributes to the pathogenesis of AD and DS. We determined that full-length amyloid precursor protein (APP) and its ß-C-terminal fragment (ß-CTF) act though increased activation of Rab5 to cause enlargement of early endosomes and to disrupt retrograde axonal trafficking of nerve growth factor (NGF) signals. The functional impacts of APP and its various products were investigated in PC12 cells, cultured rat basal forebrain cholinergic neurons (BFCNs), and BFCNs from a mouse model of DS. We found that the full-length wild-type APP (APPWT) and ß-CTF both induced endosomal enlargement and disrupted NGF signaling and axonal trafficking. ß-CTF alone induced atrophy of BFCNs that was rescued by the dominant-negative Rab5 mutant, Rab5S34N. Moreover, expression of a dominant-negative Rab5 construct markedly reduced APP-induced axonal blockage in Drosophila. Therefore, increased APP and/or ß-CTF impact the endocytic pathway to disrupt NGF trafficking and signaling, resulting in trophic deficits in BFCNs. Our data strongly support the emerging concept that dysregulation of Rab5 activity contributes importantly to early pathogenesis of AD and DS.

Development and application of multiple immunofluorescence staining for diagnostic cytology of canine and feline lymphoma.

BACKGROUND: Immunophenotyping of canine and feline lymphoma to determine B-cell or T-cell origin is important for predicting prognosis and for development of treatment protocols. For advanced diagnostic cytology tests that can be performed on smears are required to predict the immunophenotype of lymphomas. OBJECTIVES: The aim of this study was to develop a multiple immunofluorescence (MIF) staining method for the determination of lymphocyte immunophenotype in cytologic specimens, and to evaluate its clinical utility. METHODS: B cells and T cells were detected using anti-CD79α and anti-CD3 antibodies, respectively, followed by specific fluorescence-labeled secondary antibodies. The MIF staining method was first developed using fresh-frozen sections of normal canine lymph nodes. The optimal fixative, the necessity of antigen retrieval (AR), and the optimal concentration of the antibodies were determined. The MIF method was then applied to smears of normal lymph nodes, and to clinical samples from dogs and cats with lymphoma. The MIF results were compared to genetic clonality results. RESULTS: B and T cells were detected based on specific fluorescence in frozen sections, using formalin fixation without AR. Specific fluorescence was also detected in smears from normal lymph nodes and lymphomas, and the immunophenotypes predicted from this MIF staining method completely corresponded to those from genetic clonality analysis. CONCLUSIONS: The MIF staining method that we developed in this study effectively distinguished lymphocyte immunophenotypes with high specificity and sensitivity using a single smear sample, and was useful as a diagnostic tool for canine and feline lymphoma.

Intrarenal distributions and changes of Angiotensin-converting enzyme and Angiotensin-converting enzyme 2 in feline and canine chronic kidney disease.

Angiotensin-converting enzyme (ACE) is a key enzyme in the renin-angiotensin system (RAS). ACE2 is a newly identified member of the RAS. The present immunohistochemical study focused on changes in intrarenal ACE and ACE2 immunoreactivity in feline and canine chronic kidney disease (CKD). ACE immunoreactivity was predominantly observed in the brush border of the proximal tubules in dogs and cats. ACE immunoreactivity was lower in CKD kidneys than in normal kidneys, and quantitative analysis demonstrated negative correlations between ACE and renal tissue damage in dogs. ACE2 immunoreactivity was also detected in the proximal tubules; it increased or decreased with CKD in dogs, depending on the renal region assessed. The changes in ACE and ACE2 in CKD were associated with the plasma creatinine concentration in dogs. Findings from dogs with glomerulonephritis were similar to those from dogs with non-glomerulonephritis. The present study suggests that changes in the intrarenal expression of ACE and ACE2 contribute to the pathological mechanisms of canine CKD, but not to the mechanisms of feline CKD.

Identification of small molecule activators of cryptochrome.

Impairment of the circadian clock has been associated with numerous disorders, including metabolic disease. Although small molecules that modulate clock function might offer therapeutic approaches to such diseases, only a few compounds have been identified that selectively target core clock proteins. From an unbiased cell-based circadian phenotypic screen, we identified KL001, a small molecule that specifically interacts with cryptochrome (CRY). KL001 prevented ubiquitin-dependent degradation of CRY, resulting in lengthening of the circadian period. In combination with mathematical modeling, our studies using KL001 revealed that CRY1 and CRY2 share a similar functional role in the period regulation. Furthermore, KL001-mediated CRY stabilization inhibited glucagon-induced gluconeogenesis in primary hepatocytes. KL001 thus provides a tool to study the regulation of CRY-dependent physiology and aid development of clock-based therapeutics of diabetes.