Discovery of small molecule benzothiazole and indole derivatives tackling tau 2N4R and α-synuclein fibrils.

Tau and α-synuclein aggregates are the main histopathological hallmarks present in Alzheimer's disease (AD), Parkinson's disease (PD), and other neurodegenerative disorders. Intraneuronal hyperphosphorylated tau accumulation is significantly connected to the degree of cognitive impairment in AD patients. In particular, the longest 2N4R tau isoform has a propensity to rapidly form oligomers and mature fibrils. On the other hand, misfolding of α-synuclein (α-syn) is the characteristic feature in PD and dementia with Lewy bodies (DLB). There is a strong crosstalk between the two prone-to-aggregation proteins as they coprecipitated in some brains of AD, PD, and DLB patients. Simultaneous targeting of both proteinaceous oligomers and aggregates is still challenging. Here, we rationally designed and synthesized benzothiazole- and indole-based compounds using the structural hybridization strategy between the benzothiazole N744 cyanine dye and the diphenyl pyrazole Anle138b that showed anti-aggregation activity towards 2N4R tau and α-syn, respectively. The anti-aggregation effect of the prepared compounds was monitored using the thioflavin-T (ThT) fluorescence assay, while transmission electron microscopy (TEM) was employed to detect fibrils upon the completion of a time-course study with the ThT assay. Moreover, the photo-induced crosslinking of unmodified protein (PICUP) assay was used to determine the formation of oligomers. Specifically, compounds 46 and 48 demonstrated the highest anti-aggregation activity by decreasing the ThT fluorescence to 4.0 and 14.8%, respectively, against α-syn. Although no noticeable effect on 2N4R tau oligomers, 46 showed promising anti-oligomer activity against α-syn. Both compounds induced a significantly high anti-aggregation effect against the two protein fibrils as visualized by TEM. Moreover, compound 48 remarkably inhibited α-syn inclusion and cell confluence using M17D cells. Collectively, compounds 46 and 48 could serve as a basic structure for further optimization to develop clinically active AD and PD disease-modifying agents.

1,4-Diurea- and 1,4-Dithiourea-Substituted Aromatic Derivatives Selectively Inhibit α-Synuclein Oligomer Formation In Vitro.

Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting the elderly population worldwide. In PD, the misfolding of α-synuclein (α-syn) results in the formation of inclusions referred to as Lewy bodies (LB) in midbrain neurons of the substantia nigra and other specific brain localizations, which is associated with neurodegeneration. There are no approved strategies to reduce the formation of LB in the neurons of patients with PD. Our drug discovery program focuses on the synthesis of urea and thiourea compounds coupled with aminoindole moieties to abrogate α-syn aggregation and to slow down the progression of PD. We synthesized several urea and thiourea analogues with a central 1,4-phenyl diurea/thiourea linkage and evaluated their effectiveness in reducing α-syn aggregation with a special focus on the selective inhibition of oligomer formation among other proteins. We utilized biophysical methods such as thioflavin T (ThT) fluorescence assays, transmission electron microscopy (TEM), photoinduced cross-linking of unmodified proteins (PICUP), as well as M17D intracellular inclusion cell-based assays to evaluate the antiaggregation properties and cellular protection of our best compounds. Our results identified compound 1 as the best compound in reducing α-syn fibril formation via ThT assays. The antioligomer formation of compound 1 was subsequently superseded by compound 2. Both compounds selectively curtailed the oligomer formation of α-syn but not tau 4R isoforms (0N4R, 2N4R) or p-tau (isoform 1N4R). Compounds 1 and 2 failed to abrogate tau 0N3R fibril formation by ThT and atomic force microscopy. Compound 2 was best at reducing the formation of recombinant α-syn fibrils by TEM. In contrast to compound 2, compound 1 reduced the formation of α-syn inclusions in M17D neuroblastoma cells in a dose-dependent manner. Compound 1 may provide molecular scaffolds for the optimization of symmetric molecules for its α-syn antiaggregation activity with potential therapeutic applications and development of small molecules in PD.

Lumbar vertebral bone density is decreased in horses with pituitary pars intermedia dysfunction.

BACKGROUND: Pathological fractures have been reported in equids with pituitary pars intermedia dysfunction (PPID) but their prevalence and pathogenesis is unknown. OBJECTIVES: To compare: (1) bone mineral density (BMD) in weight bearing and nonweight bearing bones in PPID+ equids and aged and young PPID- controls; and (2) biomechanical properties of the fourth lumbar vertebral body in PPID+ equids and aged PPID- equids. STUDY DESIGN: Case-control study: five PPID+ equids and six aged and four young PPID- control horses. METHODS: PPID status was based on clinical signs and necropsy examination of the pituitary gland (PG). The lumbar vertebral column, right front third metacarpus (MC3), left hind third metatarsus (MT3), and PG were removed after euthanasia. BMD was determined by quantitative computed tomography of regions of interest (ROI) in each bone and biomechanical testing was performed on the fourth lumbar vertebral body. Serum concentrations of parathormone (PTH), ionised Ca++ , 25-hydroxyvitamin D, and osteocalcin (OC) were also measured. Data were analysed using one-way ANOVA and correlation analyses. RESULTS: BMD of trabecular and cortical regions of interest (ROI) of the third, fourth (L4), and fifth lumbar vertebrae were significantly lower in PPID+ equids as compared with aged (p < 0. 001) and young (p < 0.01) PPID- controls. In contrast, no differences were found in BMD of trabecular or cortical ROIs of MC3 and MT3 between groups. No differences were detected in force at fracture, displacement at fracture, Young's modulus or strain of L4 between PPID+ and aged PPID- horses. No differences were found in serum PTH, ionised Ca++ , 25-hydroxyvitamin D, or OC concentrations between groups. MAIN LIMITATIONS: Limited number of equids studied and variation in test results. CONCLUSIONS: BMD of nonweight bearing bones can be decreased with PPID and could increase risk of developing pathological fractures.

Investigation of serum amyloid a within animal species focusing on the 1-25 amino acid region.

AA amyloidosis, characterized by the misfolding of serum amyloid A (SAA) protein, is the most common amyloid protein disorder across multiple species. SAA is a positive-acute phase protein synthesized by the liver in response to inflammation or stress, and it normally associates with high-density lipoprotein at its N-terminus. In this study, we focused on the 1-25 amino acid (aa) region of the complete 104 aa SAA sequence to examine the aggregation propensity of AA amyloid. A library comprising eight peptides from different species was assembled for analysis. To access the aggregation propensity of each peptide region, a bioinformatic study was conducted using the algorithm TANGO. Congo red (CR) binding assays, Thioflavin T (ThT) assays, and transmission electron microscopy (TEM) were utilized to evaluate whether the synthesized peptides formed amyloid-like fibrils. All synthetic SAA 1-25 congeners resulted in amyloid-like fibrils formation (per CR and/or ThT staining and TEM detection) at the exception of the ferret SAA1-25 fragment, which generated plaque-like materials by TEM. Ten residues were preserved among SAA 1-25 congeners resulting in amyloid-like fibrils, i.e. F6, E9, A10, G13, D16, M17, A20, Y21, D23, and M24. Amino acid residues highlighted by this study may have a role in increasing the propensity for amyloid-like fibril formation. This study put an emphasis on region 1-25 in the mechanism of SAA1 misfolding.

Evaluation of N- and O-Linked Indole Triazines for a Dual Effect on α-Synuclein and Tau Aggregation.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder underlying dementia in the geriatric population. AD manifests by two pathological hallmarks: extracellular amyloid-ß (Aß) peptide-containing senile plaques and intraneuronal neurofibrillary tangles comprised of aggregated hyperphosphorylated tau protein (p-tau). However, more than half of AD cases also display the presence of aggregated α-synuclein (α-syn)-containing Lewy bodies. Conversely, Lewy bodies disorders have been reported to have concomitant Aß plaques and neurofibrillary tangles. Our drug discovery program focuses on the synthesis of multitarget-directed ligands to abrogate aberrant α-syn, tau (2N4R), and p-tau (1N4R) aggregation and to slow the progression of AD and related dementias. To this end, we synthesized 11 compounds with a triazine-linker and evaluated their effectiveness in reducing α-syn, tau isoform 2N4R, and p-tau isoform 1N4R aggregation. We utilized biophysical methods such as thioflavin T (ThT) fluorescence assays, transmission electron microscopy (TEM), photoinduced cross-linking of unmodified proteins (PICUP), and M17D intracellular inclusion cell-based assays to evaluate the antiaggregation properties and cellular protection of our best compounds. We also performed disaggregation assays with isolated Aß-plaques from human AD brains. Our results demonstrated that compound 10 was effective in reducing both oligomerization and fibril formation of α-syn and tau isoform 2N4R in a dose-dependent manner via ThT and PICUP assays. Compound 10 was also effective at reducing the formation of recombinant α-syn, tau 2N4R, and p-tau 1N4R fibrils by TEM. Compound 10 reduced the development of α-syn inclusions in M17D neuroblastoma cells and stopped the seeding of tau P301S using biosensor cells. Disaggregation experiments showed smaller Aß-plaques and less paired helical filaments with compound 10. Compound 10 may provide molecular scaffolds for further optimization and preclinical studies for neurodegenerative proteinopathies.

Diabetes mellitus drug discovery: insights into targeting feline and human amylin with small molecules.

BACKGROUND: Type 2 diabetes (T2D) is a health concern for both humans and cats, with cases rising over the past decade. Around 70% of patients from either species exhibit pancreatic aggregates of islet amyloid polypeptide (IAPP), a protein that proves toxic upon misfolding. These misfolded protein aggregates congregate in the islets of Langerhans of the pancreas, diminishing the capability of ß-cells to produce insulin and further perpetuating disease. OBJECTIVE: Our team's drug discovery program is investigating newly synthesized compounds that could diminish aggregates of both human and feline IAPP, potentially disrupting the progression of T2D. MATERIAL AND METHODS: We prepared 24 compounds derived from diaryl urea, as ureas have previously demonstrated great potential at reducing accumulations of misfolded proteins. Biophysical methods were employed to analyze the anti-aggregation activity of these compounds at inhibiting and/or disrupting IAPP fibril formation in vitro. RESULTS: The results demonstrate that compounds 12 and 24 were most effective at reducing the fibrillization and aggregation of both human and feline IAPP. When compared with the control for each experiment, samples treated with either compound 12 or 24 exhibited fewer accumulations of amyloid-like fibrils. CONCLUSION: Urea-based compounds, such as compounds 12 and 24, may prove crucial in future pre-clinical studies in the search for therapeutics for T2D.

Discovery of 4-aminoindole carboxamide derivatives to curtail alpha-synuclein and tau isoform 2N4R oligomer formation.

Alzheimer's disease (AD) is a multifactorial, chronic neurodegenerative disease characterized by the presence of extracellular ß-amyloid (Aß) plaques, intraneuronal neurofibrillary tangles (NFTs), activated microglial cells, and an inflammatory state (involving reactive oxygen species production) in the brain. NFTs are comprised of misfolded and hyperphosphorylated forms of the microtubule-binding protein tau. Interestingly, the trimeric form of the 2N4R splice isoform of tau has been found to be more toxic than the trimeric 1N4R isoform in neuron precursor cells. Few drug discovery programs have focused on specific tau isoforms. The present drug discovery project is centered on the anti-aggregation effect of a series of seventeen 4- or 5-aminoindole carboxamides on the 2N4R isoform of tau. The selection of the best compounds was performed using α-synuclein (α-syn). The anti-oligomer and -fibril activities of newly synthesized aminoindole carboxamide derivatives were evaluated with biophysical methods, such as thioflavin T fluorescence assays, photo-induced cross-linking of unmodified proteins, and transmission electron microscopy. To evaluate the reduction of inclusions and cytoprotective effects, M17D neuroblastoma cells expressing inclusion-forming α-syn were treated with the best amide representatives. The 4-aminoindole carboxamide derivatives exhibited a better anti-fibrillar activity compared to their 5-aminoindole counterparts. The amide derivatives 2, 8, and 17 exerted anti-oligomer and anti-fibril activities on α-syn and the 2N4R isoform of tau. At a concentration of 40 µM, compound 8 reduced inclusion formation in M17D neuroblastoma cells expressing inclusion-prone αSynuclein3K::YFP. Our results demonstrate the potential of 4-aminoindole carboxamide derivatives with regard to inhibiting the oligomer formation of α-syn and tau (2N4R isoform) for further optimization prior to pre-clinical studies.

5-Nitro-1,2-benzothiazol-3-amine and N-Ethyl-1-[(ethylcarbamoyl)(5-nitro-1,2-benzothiazol-3-yl)amino]formamide Modulate α-Synuclein and Tau Aggregation.

Protein misfolding results in a plethora of known diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, transthyretin-related amyloidosis, type 2 diabetes, Lewy body dementia, and spongiform encephalopathy. To provide a diverse portfolio of therapeutic small molecules with the ability to reduce protein misfolding, we evaluated a set of 13 compounds: 4-(benzo[d]thiazol-2-yl)aniline (BTA) and its derivatives containing urea (1), thiourea (2), sulfonamide (3), triazole (4), and triazine (5) linker. In addition, we explored small modifications on a very potent antioligomer 5-nitro-1,2-benzothiazol-3-amine (5-NBA) (compounds 6-13). This study aims to define the activity of BTA and its derivatives on a variety of prone-to-aggregate proteins such as transthyretin (TTR81-127, TTR101-125), α-synuclein (α-syn), and tau isoform 2N4R (tau 2N4R) through various biophysical methods. Thioflavin T (ThT) fluorescence assay was used to monitor fibril formation of the previously mentioned proteins after treatment with BTA and its derivatives. Antifibrillary activity was confirmed using transmission electron microscopy (TEM). Photoreactive cross-linking assay (PICUP) was utilized to detect antioligomer activity and lead to the identification of 5-NBA (at low micromolar concentration) and compound 13 (at high concentration) as the most promising in reducing oligomerization. 5-NBA and not BTA inhibited the inclusion formation based on the cell-based assay using M17D neuroblastoma cells that express inclusion-prone αS-3K::YFP. 5-NBA abrogated the fibril, oligomer, and inclusion formation in a dose-dependent manner. 5-NBA derivatives could be the key to mitigate protein aggregation. In the future, the results made from this study will provide an initial platform to generate more potent inhibitors of α-syn and tau 2N4R oligomer and fibril formation.

Hyperphosphorylated tau (p-tau) and drug discovery in the context of Alzheimer's disease and related tauopathies.

Alzheimer's disease (AD) is the most common form of dementia, characterized by intracellular neurofibrillary tangles (NFTs) and extracellular ß-amyloid (ßA) plaques. No disease-modifying therapy is currently available to prevent the progression of, or cure, the disease. Misfolded hyperphosphorylated tau (p-tau) is considered a pivotal point in the pathogenesis of AD and other tauopathies. Compelling evidence suggests that it is a key driver of the accumulation of NFTs and can be directly correlated with the extent of dementia in patients with AD. Therefore, inhibiting tau hyperphosphorylation-induced aggregation could be a viable strategy to discover and develop therapeutics for patients with AD.

Anti-fibrillization effects of sulfonamide derivatives on α-synuclein and hyperphosphorylated tau isoform 1N4R.

In contrast to Aß plaques, the spatiotemporal distribution of neurofibrillary tangles of hyperphosphorylated tau (p-tau) predicts cognitive impairment in Alzheimer's disease (AD), underscoring the key pathological role of p-tau and the utmost need to develop AD therapeutics centering upon the control of p-tau aggregation and cytotoxicity. Our drug discovery program is focused on compounds that prevent the aggregation and cytotoxicity of p-tau moieties of the tau isoform 1N4R due to its prevalence (1 N) and long-distance trans-synaptic propagation (4R). We prepared and tested twenty-four newly synthesized small molecules representing the urea (1, 2, 3), sulfonylurea (4), and sulfonamide (5-24) series and evaluated their anti-aggregation effects with biophysical methods (thioflavin T and S fluorescence assays, transmission electron microscopy) and intracellular inclusion cell-based assays. Pre-evaluation was performed on alpha-synuclein (α-syn) to identify molecules to be challenged with p-tau. The sulfonamide derivatives 18 and 20 exhibited an anti-fribrillization activity on α-syn and p-tau. Sulfonamide compounds 18 and 20 reduced inclusion formation in M17D neuroblastoma cells that express inclusion-prone αSynuclein3K::YFP. This project advances new concepts in targeting prone-to-aggregate proteins such as α-syn and p-tau, and provides a molecular scaffold for further optimization and pre-clinical studies focused on AD drug development.

Recent Advances in the Discovery of Therapeutics to Curtail Islet Amyloid Polypeptide Aggregation for Type 2 Diabetes Treatment.

In humans with type 2 diabetes, at least 70% of patients exhibit islet amyloid plaques formed by misfolding islet amyloid polypeptides (IAPP). The oligomeric conformation and accumulation of the IAPP plaques lead to a panoply of cytotoxic effects on the islet ß-cells. Currently, no marketed therapies for the prevention or elimination of these amyloid deposits exist, and therefore significant efforts are required to address this gap. To date, most of the experimental treatments are limited to only in vitro stages of testing. In general, the proposed therapeutics use various targeting strategies, such as binding to the N-terminal region of islet amyloid polypeptide on residues 1-19 or the hydrophobic region of IAPP. Other strategies include targeting the peptide self-assembly through π-stacking. These methods are realized by using several different families of compounds, four of which are highlighted in this review: naturally occurring products, small molecules, organometallic compounds, and nanoparticles. Each of these categories holds immense potential to optimize and develop inhibitor(s) of pancreatic amyloidosis in the near future.

Revisiting misfolding propensity of serum amyloid A1: Special focus on the signal peptide region.

AA amyloidosis is the result of overproduction and aberrant processing of acute-phase serum amyloid A1 (SAA1) by hepatocytes. Proteolytic cleavage of SAA1 is believed to play a central role in AA amyloid formation. The SAA1 protein undergoes a cleavage of 18 residues consisting of the signal peptide at the N-terminal region. To better understand the mechanism behind systemic amyloidosis in the SAA1 protein, we studied the misfolding propensity of the signal peptide region. We first examined the signal peptide amino acid SAA derived from different animal species. A library of 16 peptides was designed to evaluate the propensity of aggregation. The amyloidogenic potential of each SAA1 signal peptide homolog was assessed using in silico Tango program, thioflavin T (ThT) fluorescence, transmission electron microscopy (TEM), and seeding with misfolded human SAA1 signal peptide. After 7 days of incubation, most of the SAA1 signal peptide fragments had the propensity to form fibrils at a concentration of 100 µM in 50 mM Tris buffer at 37 °C by TEM. All peptides were able to generate fibrils at a higher concentration, i.e 500 µM in 25 mM Tris buffer with 50% HFIP, by ThT. All SAA1 signal synthetic peptides designed from the different animal species had the propensity to misfold and form fibrils, particularly in species with low occurrence of systemic amyloidosis. The human SAA1 signal peptide region was capable to seed the SAA1 1-25 and 32-47 peptide regions. Characterizing fibrillar conformations are relevant for seeding intact and/or fragmented SAA, which may contribute, to the mechanism of protein misfolding. This research signifies the importance of the signal peptide region and its possible contribution to the misfolding of aggregation-prone proteins.

Equine pituitary pars intermedia dysfunction: a spontaneous model of synucleinopathy.

Equine pituitary pars intermedia dysfunction (PPID) is a common endocrine disease of aged horses that shows a similar pathophysiology as Parkinson's Disease (PD) with increased levels of α-synuclein (α-syn). While α-syn is thought to play a pathogenic role in horses with PPID, it is unclear if α-syn is also misfolded in the pars intermedia and could similarly promote self-aggregation and propagation. Consequently, α-syn was isolated from the pars intermedia from groups of healthy young and aged horses, and aged PPID-afflicted horses. Seeding experiments confirmed the prion-like properties of α-syn isolated from PPID-afflicted horses. Next, detection of α-syn fibrils in pars intermedia via transmission electron microscopy (TEM) was exclusive to PPID-afflicted horses. A bank of fragment peptides was designed to further characterize equine α-syn misfolding. Region 62-87 of equine and human α-syn peptides was found to be most prone to aggregation according to Tango bioinformatic program and kinetics of aggregation via a thioflavin T fluorescence assay. In both species, fragment peptide 62-87 is capable of generating mature fibrils as demonstrated by TEM. The combined animal, bioinformatic, and biophysical studies provide evidence that equine α-syn is misfolded in PPID horses.

Survey of amyloidosis cases among different free-living wild and zoo animals.

Amyloidosis comprises a range of protein-folding disorders characterised by a buildup of amyloid deposits in one or multiple organs. The pathogenesis and pathologic findings of amyloidosis can vary widely due to the nature of the precursor protein. In veterinary medicine, there are 10 proteins known to form amyloid deposits in various organs. This review aims to compare amyloidosis cases among different free-living wild and zoo animals focussing in part on the determination of the species particularly susceptible to the amyloid formation and specific prone-to-aggregate protein commonly involved. This review addresses the transmission of AA amyloidosis pertinent to institutions, such as zoos, housing multiple individuals and species in relatively close proximity. In addition, this review includes summarisation for definitive diagnosis of single or multiple cases of amyloidosis affecting free-living wild and zoo animals. Insights into the diversity, transmission, and pathogenesis of known amyloidogenic proteins and species prevalently affected may help to establish a preventive intervention and stimulate the discovery of new diagnostic and therapeutic strategies.

Widespread severe myodegeneration in a compound heterozygote female dog with dystrophin deficiency.

The University of Missouri (MU) has established a colony of dystrophin-deficient dogs with a mixed breed background to mirror the variable pathologic effects of dystrophinopathies between persons of a given kindred to further the understanding of the genetic and molecular basis of the variable phenotype; thus to facilitate discovery of an effective therapeutic strategy. Herein we report the phenotype and genotype of a normal-appearing 10-month-old colony female that died suddenly. At necropsy examination, there were reduced skeletal and laryngeal muscle volume and mild dilatation of the oesophagus. Microscopic findings consisted of extensive degeneration and regeneration of the axial skeletal, tongue, oesophageal, and laryngeal muscles that were characterized by considerable central nucleation, individual fibre mineralization and interstitial fibrosis. The myocardial findings were limited to infiltration of adipose cells in the interstitium. The female dog was a compound heterozygote with one X chromosome carrying a point mutation in intron 6 of the dystrophin gene and the other X chromosome carrying a repetitive element insertion in intron 13 of the dystrophin gene. Although the direct cause of death was uncertain, it might likely be due to sudden cardiac death as has been seen in Duchenne muscular dystrophy patients. This case demonstrated dystrophinopathy in female dogs that have no ameliorating normal X chromosome.

Impact of Preanalytical Factors During Histology Processing on Section Suitability for Digital Image Analysis.

Digital image analysis (DIA) is impacted by the quality of tissue staining. This study examined the influence of preanalytical variables-staining protocol design, reagent quality, section attributes, and instrumentation-on the performance of automated DIA software. Our hypotheses were that (1) staining intensity is impacted by subtle differences in protocol design, reagent quality, and section composition and that (2) identically programmed and loaded stainers will produce equivalent immunohistochemical (IHC) staining. We tested these propositions by using 1 hematoxylin and eosin stainer to process 13 formalin-fixed, paraffin-embedded (FFPE) mouse tissues and by using 3 identically programmed and loaded immunostainers to process 5 FFPE mouse tissues for 4 cell biomarkers. Digital images of stained sections acquired with a commercial whole slide scanner were analyzed by customizable algorithms incorporated into commercially available DIA software. Staining intensity as viewed qualitatively by an observer and/or quantitatively by DIA was affected by staining conditions and tissue attributes. Intrarun and inter-run IHC staining intensities were equivalent for each tissue when processed on a given stainer but varied measurably across stainers. Our data indicate that staining quality must be monitored for each method and stainer to ensure that preanalytical factors do not impact digital pathology data quality.

Nephroblastoma in a Sprague Dawley rat unrelated to titanium dioxide nanoparticle exposure in utero.

Nephroblastoma is an embryonal tumour that has rarely been reported in laboratory rats. In this case report, a large nephroblastoma with peritoneal seeding was found during necropsy in an 11-month-old, female, Sprague Dawley rat. The rat had a history of indirect exposure to nano-TiO2 (titanium dioxide nanoparticles) during maternal gestation. A firm mass in the upper right abdominal quadrant was palpated. Four weeks later, the animal quickly declined. Nephroblastoma was confirmed by histopathology. Only one rat developed nephroblastoma among the ten littermates. Nephroblastomas in Sprague Dawley rats are typically spontaneous tumours with non-malignant mesenchymal elements. The capability to induce a nephroblastoma with nano-TiO2 is less likely in this case.

Restoring pars intermedia dopamine concentrations and tyrosine hydroxylase expression levels with pergolide: evidence from horses with pituitary pars intermedia dysfunction.

BACKGROUND: Pituitary pars intermedia dysfunction (PPID) develops slowly in aged horses as degeneration of hypothalamic dopaminergic neurons leads to proliferation of pars intermedia (PI) melanotropes through hyperplasia and adenoma formation. Dopamine (DA) concentrations and tyrosine hydroxylase (TH) immunoreactivity are markedly reduced in PI tissue of PPID-affected equids and treatment with the DA receptor agonist pergolide results in notable clinical improvement. Thus, we hypothesized that pergolide treatment of PPID-affected horses would result in greater DA and TH levels in PI tissue collected from PPID-affected horses versus untreated PPID-affected horses. To test this hypothesis, pituitary glands were removed from 18 horses: four untreated PPID-affected horses, four aged and four young horses without signs of PPID, and six PPID-affected horses that had been treated with pergolide at 2 µg/kg orally once daily for 6 months. DA concentrations and TH expression levels in PI tissues were determined by high performance liquid chromatography with electrochemical detection and Western blot analyses, respectively. RESULTS: DA and TH levels were lowest in PI collected from untreated PPID-affected horses while levels in the pergolide treated horses were similar to those of aged horses without signs of PPID. CONCLUSIONS: These findings provide evidence of restoration of DA and TH levels following treatment with pergolide. Equine PPID is a potential animal model of dopaminergic neurodegeneration, which could provide insight into human neurodegenerative diseases.

Hyperphosphorylation Renders Tau Prone to Aggregate and to Cause Cell Death.

Alzheimer's disease (AD) is a neurodegenerative disorder without a cure or prevention to date. Hyperphosphorylated tau forms the neurofibrillary tangles (NFTs) that correlate well with the progression of cognitive impairments. Animal studies demonstrated the pathogenic role of hyperphosphorylated tau. Understanding how abnormal phosphorylation renders a normal tau prone to form toxic fibrils is key to delineating molecular pathology and to developing efficacious drugs for AD. Production of a tau bearing the disease-relevant hyperphosphorylation and molecular characters is a pivotal step. Here, we report the preparation and characterization of a recombinant hyperphosphorylated tau (p-tau) with strong relevance to disease. P-tau generated by the PIMAX approach resulted in phosphorylation at multiple epitopes linked to the progression of AD neuropathology. In stark contrast to unmodified tau that required an aggregation inducer, and which had minimal effects on cell functions, p-tau formed inducer-free fibrils that triggered a spike of mitochondrial superoxide, induced apoptosis, and caused cell death at sub-micromolar concentrations. P-tau-induced apoptosis was suppressed by inhibitors for reactive oxygen species. Hyperphosphorylation apparently caused rapid formation of a disease-related conformation. In both aggregation and cytotoxicity, p-tau exhibited seeding activities that converted the unmodified tau into a cytotoxic species with an increased propensity for fibrillization. These characters of p-tau are consistent with the emerging view that hyperphosphorylation causes tau to become an aggregation-prone and cytotoxic species that underlies diffusible pathology in AD and other tauopathies. Our results further suggest that p-tau affords a feasible tool for Alzheimer's disease mechanistic and drug discovery studies.