Does modulation of tau hyperphosphorylation represent a reasonable therapeutic strategy for Alzheimer's disease? From preclinical studies to the clinical trials.

Protein kinases (PKs) have emerged as one of the most intensively investigated drug targets in current pharmacological research, with indications ranging from oncology to neurodegeneration. Tau protein hyperphosphorylation was the first pathological post-translational modification of tau protein described in Alzheimer's disease (AD), highlighting the role of PKs in neurodegeneration. The therapeutic potential of protein kinase inhibitors (PKIs)) and protein phosphatase 2 A (PP2A) activators in AD has recently been explored in several preclinical and clinical studies with variable outcomes. Where a number of preclinical studies demonstrate a visible reduction in the levels of phospho-tau in transgenic tauopathy models, no reduction in neurofibrillary lesions is observed. Amongst the few PKIs and PP2A activators that progressed to clinical trials, most failed on the efficacy front, with only a few still unconfirmed and potential positive trends. This suggests that robust preclinical and clinical data is needed to unequivocally evaluate their efficacy. To this end, we take a systematic look at the results of preclinical and clinical studies of PKIs and PP2A activators, and the evidence they provide regarding the utility of this approach to evaluate the potential of targeting tau hyperphosphorylation as a disease modifying therapy.

Plasma Leptin Reflects Progression of Neurofibrillary Pathology in Animal Model of Tauopathy.

The close relationship between Alzheimer's disease (AD) and obesity was recognized many years ago. However, complete understanding of the pathological mechanisms underlying the interactions between degeneration of CNS and fat metabolism is still missing. The leptin a key adipokine of white adipose tissue has been suggested as one of the major mediators linking the obesity and AD. Here we investigated the association between peripheral levels of leptin, general metabolic status and stage of the pathogenesis in rat transgenic model of AD. We demonstrate significantly decreased levels of plasma leptin in animals with experimentally induced progressive neurofibrillary pathology, which represents only 62.3% (P = 0.0015) of those observed in normal wild type control animals. More detailed analysis showed a strong and statistically significant inverse correlation between the load of neurofibrillary pathology and peripheral levels of leptin (r = - 0.7248, P = 0.0177). We also observed a loss of body weight during development of neurodegeneration (about 14% less than control animals, P = 0.0004) and decrease in several metabolic parameters such as glucose, insulin, triglycerides and VLDL in plasma of the transgenic animals. Our data suggest that plasma leptin could serve as a convenient peripheral biomarker for tauopathies and Alzheimer's disease. Decrease in gene expression of leptin in fat tissue and its plasma level was found as one of the consequences of experimentally induced neurodegeneration. Our data may help to design rational diagnostic and therapeutic strategies for patients suffering from Alzheimer's disease or other forms of tauopathy.

Transcriptomic signature of Alzheimer's disease tau seed-induced pathology.

Spreading of tau pathology to anatomical distinct regions in Alzheimer's disease (AD) is associated with progression of the disease. Studies in recent decade have strived to understand the processes involved in this characteristic spread. We recently showed that AD-derived insoluble tau seeds are able to initiate neurofibrillary pathology in transgenic rodent model of tauopathy. In the present study, we pursued to identify the molecular changes that govern the induction and propagation of tau pathology on the transcriptomic level. We first show that microglia in vicinity to AD-Tau-induced pathology has phagocytic morphology when compared to PBS-injected group. On transcriptomic level, we observed deregulation of 15 genes 3-month post AD-Tau seeds inoculation. Integrated bioinformatic analysis identified 31 significantly enriched pathways. Amongst these, the inflammatory signalling pathway mediated by cytokine and chemokine networks, along with, toll-like receptor and JAK-STAT signalling were the most dominant. Furthermore, the enriched signalling also involved the regulation of autophagy, mitophagy and endoplasmic reticulum stress pathways. To our best of knowledge, the study is the first to investigate the transcriptomic profile of AD-Tau seed-induced pathology in hippocampus of transgenic model of tauopathy.

Hypertension does not alter disturbances in leptin signalling observed in experimental model of tauopathy.

Neurodegeneration is associated with hypertension and disturbance in fat metabolism. The complex interaction of neurodegenerative processes with both metabolic changes and blood pressure is still not fully elucidated. Here we demonstrate that the experimentally induced tauopathy in hypertensive transgenic animals causes significant downregulation of plasma leptin (53% of control), reduction of body weight by 11%, a 1.2-fold drop of adiposity index, and decrease in HDL cholesterol level, while the fasting glucose and insulin concentration remain unchanged. Despite of these alterations we found the leptin projection circuit including the arcuate nucleus, paraventricular nucleus in hypothalamus, and nucleus tractus solitarius in the brainstem not affected by neurofibrillary pathology. Furthermore, hypertension does not alter disturbances in leptin signalling. The presented data provide further insight into neurodegeneration-induced metabolic alterations relevant for human tauopathies.

Improved tissue integrity after alginate treatment in rat spinal cord injury: evidence from ex vivo diffusion tensor imaging.

Diffusion tensor imaging (DTI) is a magnetic resonance imaging technique used to characterize fibrous structures such as white matter in the central nervous system, including normal and spinal cord injury (SCI) conditions. Our aim was to evaluate the effect of alginate treatment in the rat SCI by DTI parametric measures. Ex vivo DTI data were collected by spin echo sequence with following parameters TR/TE: 2500 ms/32 ms and b-value of 1500 s/mm2. Main significant changes were found in fractional anisotropy (FA), and radial diffusivity (RD), between the saline- and alginatetreated group at the level of individual sections and whole spinal cord. Results indicate that ex vivo DTI can be used as a tool for tissue structure characterisation and both FA and RD as promising prognostic parameters of SCI treatment.

Diffusion Tensor Imaging: Tool for Tracking Injured Spinal Cord Fibres in Rat.

Spinal cord injury (SCI) is a severe disorder of the CNS leading to tissue damage and disability. Because it is critical to understand the pathological processes, it is important to find efficient ways to diagnose the severity of injured spinal cord tracts in situ from beginning up to a certain level of recovery following therapeutic interventions. In the current study, we set-up the criteria for diffusion tensor imaging (DTI) in order to capture changes of nerve fibre tracts in rat spinal cord compression injury. We tested four DTI parameters, such as fractional anisotropy, mean diffusivity, axial diffusivity and radial diffusivity at the lesion site, in time course of 7 weeks. Afterwards, we compared DTI data with histological results and locomotor outcomes to examine their consistency and capability of reflecting the lesion development in time. Our data confirm that DTI is a valuable in vivo imaging tool capable to distinguish damaged white matter tracts after mild SCI in rat. Fractional anisotropy showed decreased values for injury site, while the mean diffusivity had higher values, with increased both axial and radial diffusivity in comparison to control subjects. Thus, the combination of DTI parameters can reflect the severity of lesion in time and may correlate with histological evaluation of spared tissue, but not with locomotor recovery following mild injury associated with spontaneous recovery.

Neuropathology and biochemistry of early onset familial Alzheimer's disease caused by presenilin-1 missense mutation Thr116Asn.

The majority (~ 55%) of early onset familial Alzheimer disease (FAD) is caused by mutations in the presenilin 1 gene (PSEN1). Here, we describe a family with early onset FAD with a missense mutation in the PSEN1 gene (Thr116Asn). Five family members developed dementia in the third decade of life. One subject underwent autopsy. The onset of clinical symptoms was at the age of 37 years and the disease progressed rapidly. The clinical picture was characterised by progressive memory impairment, amnestic aphasia, and gait disturbances. Neuropathological assessment revealed widespread ß-amyloid (Thal phase 5) and tau (Braak stage 6) pathology. Abundant deposition of diffuse and cored plaques was distributed in cortical and subcortical areas, as well as in the cerebellum, while cotton wool plaques were observed mainly in the occipital cortex. Cerebral amyloid angiopathy was present throughout the brain. In the neocortex, tau pathology, especially neuropil threads, was more abundant in the frontal and occipital cortex and in the hippocampus. Proteomic analyses revealed that the pattern of sarkosyl-insoluble tau was similar to the one seen in sporadic AD. No α-synuclein or TDP-43 pathology was found either in cortical nor in subcortical areas. Here, we present the first comprehensive neuropathological and biochemical study of early onset FAD with a missense mutation Thr116Asn in the presenilin 1 gene. In contrast to other PS1-linked AD patients, the present subject developed cotton wool plaques which were not associated with spastic paraparesis.

Localized Intrathecal Delivery of Mesenchymal Stromal Cells Conditioned Medium Improves Functional Recovery in a Rat Model of Spinal Cord Injury.

It was recently shown that the conditioned medium (CM) of mesenchymal stem cells can enhance viability of neural and glial cell populations. In the present study, we have investigated a cell-free approach via CM from rat bone marrow stromal cells (MScCM) applied intrathecally (IT) for spinal cord injury (SCI) recovery in adult rats. Functional in vitro test on dorsal root ganglion (DRG) primary cultures confirmed biological properties of collected MScCM for production of neurosphere-like structures and axon outgrowth. Afterwards, rats underwent SCI and were treated with IT delivery of MScCM or vehicle at postsurgical Days 1, 5, 9, and 13, and left to survive 10 weeks. Rats that received MScCM showed significantly higher motor function recovery, increase in spared spinal cord tissue, enhanced GAP-43 expression and attenuated inflammation in comparison with vehicle-treated rats. Spared tissue around the lesion site was infiltrated with GAP-43-labeled axons at four weeks that gradually decreased at 10 weeks. Finally, a cytokine array performed on spinal cord extracts after MScCM treatment revealed decreased levels of IL-2, IL-6 and TNFα when compared to vehicle group. In conclusion, our results suggest that molecular cocktail found in MScCM is favorable for final neuroregeneration after SCI.

Intraneuronal accumulation of misfolded tau protein induces overexpression of Hsp27 in activated astrocytes.

Accumulation of misfolded forms of microtubule associated, neuronal protein tau causes neurofibrillary degeneration typical of Alzheimer's disease and other tauopathies. This process is accompanied by elevated cellular stress and concomitant deregulation of heat-shock proteins. We used a transgenic rat model of tauopathy to study involvement of heat shock protein 27 (Hsp27) in the process of neurofibrillary degeneration, its cell type specific expression and correlation with the amount of insoluble tau protein aggregates. The expression of Hsp27-mRNA is more than doubled and levels of Hsp27 protein tripled in aged transgenic animals with tau pathology. The data revealed a strong positive and highly significant correlation between Hsp27-mRNA and amount of sarkosyl insoluble tau. Interestingly, intracellular accumulation of insoluble misfolded tau protein in neurons was associated with overexpression of Hsp27 almost exclusively in reactive astrocytes, not in neurons. The topological dissociation of neuronally expressed pathological tau and the induction of astrocytic Hsp27, GFAP, and Vimentin along with up-regulation of microglia specific markers such as CD18, CD68 and C3 point to cooperation of astrocytes, microglia and neurons in response to intra-neuronal accumulation of insoluble tau. Our data suggest that over expression of Hsp27 represents a part of microglia-mediated astrocytic response mechanism in the process of neurofibrillary degeneration, which is not necessarily associated with neuroprotection and which in contrary may accelerate neurodegeneration in late stage of the disease. This phenomenon should be considered during development of disease modifying strategies for treatment of tauopathies and AD via regulation of activity of Hsp27.

Atypical Huntington's disease with the clinical presentation of behavioural variant of frontotemporal dementia.

Huntington's disease is an incurable, adult-onset, autosomal dominant inherited disorder caused by an expanded trinucleotide repeat (CAG). In this study, we describe a Huntington's disease patient displaying clinical symptoms of the behavioural variant of frontotemporal dementia in the absence of tremor and ataxia. The clinical onset was at the age of 36 years and the disease progressed slowly (18 years). Genetic testing revealed expanded trinucleotide CAG repeats in the Huntingtin gene, together with a Glu318Gly polymorphism in presenilin 1. Neuropathological assessment revealed extensive amyloid ß (Aß) aggregates in all cortical regions. No inclusions displaying hyperphosphorylated tau or phosphorylated transactive response DNA-binding protein 43 (TDP43) were found. A high number of p62 (sequestosome 1) immunopositive intranuclear inclusions were seen mainly in the cortex, while subcortical areas were affected to a lesser extent. Confocal microscopy revealed that the majority of p62 intranuclear lesions co-localised with the fused-in-sarcoma protein (FUS) immunostaining. The morphology of the inclusions resembled intranuclear aggregates in Huntington's disease. The presented proband suffered from Huntington's disease showed atypical distribution of FUS positive intranuclear aggregates in the cortical areas with concomitant Alzheimer's disease pathology.

On the utility of cerebrospinal fluid biomarkers in canine neurological disorders.

The cerebral biomarkers, neurofilament light chain (NfL), amyloid-ß, tau, and neuron specific enolase (NSE) reflect a wide spectrum of neurological damage in the brain and spinal cord. With this study, we aimed to assess whether these biomarkers hold any potential diagnostic value for the three most common canine neurological diseases. Canines suffering from meningoencephalitis of unknown origin (MUO), brain tumors, and selected non-infectious myelopathies were included. For each diagnosis, we analyzed these biomarkers in the cerebrospinal fluid collected via cranial puncture from the cisterna magna. Elevated levels of CSF tau, NfL, and NSE were observed in MUO, with all three biomarkers being intercorrelated. Tau and NSE were increased while amyloid-ß was decreased in dogs suffering from tumors. In contrast, no biomarker changes were observed in dogs with myelopathies. Covariates such as age, sex, or castration had minimal impact. CSF biomarkers may reflect molecular changes related to MUO and tumors, but not to non-infectious myelopathies. The combination of NfL, tau, and NSE may represent useful biomarkers for MUO as they reflect the same pathology and are not influenced by age.

Shaping the future of preclinical development of successful disease-modifying drugs against Alzheimer's disease: a systematic review of tau propagation models.

The transcellular propagation of the aberrantly modified protein tau along the functional brain network is a key hallmark of Alzheimer's disease and related tauopathies. Inoculation-based tau propagation models can recapitulate the stereotypical spread of tau and reproduce various types of tau inclusions linked to specific tauopathy, albeit with varying degrees of fidelity. With this systematic review, we underscore the significance of judicious selection and meticulous functional, biochemical, and biophysical characterization of various tau inocula. Furthermore, we highlight the necessity of choosing suitable animal models and inoculation sites, along with the critical need for validation of fibrillary pathology using confirmatory staining, to accurately recapitulate disease-specific inclusions. As a practical guide, we put forth a framework for establishing a benchmark of inoculation-based tau propagation models that holds promise for use in preclinical testing of disease-modifying drugs.

Enriched environment ameliorates propagation of tau pathology and improves cognition in rat model of tauopathy.

Introduction: The typical symptoms of Alzheimer's disease (AD) are cognitive impairment, disrupted spatial orientation, behavioral and psychiatric abnormalities, and later motor deficits. Neuropathologically, AD is characterized by deposits of pathological forms of endogenous proteins - amyloid-ß, and neurofibrillary tau protein pathology. The latter closely correlates with brain atrophy and clinical impairment. Pharmacological therapies for these pathologies are largely absent, raising the question whether non-pharmacological interventions could be efficacious. Environmental factors can play a role in the manifestation of AD. It is unknown whether enriched environment (EE) can ameliorate the propagation of protein aggregates or their toxic components. Methods: We injected insoluble tau extracts from human brains with AD (600 or 900 ng per animal) into hippocampi of SHR72 transgenic rats that express non-mutated truncated human tau 151-391/4R, but usually do not develop hippocampal tangles. The rats had either standard housing, or could access an EE 5×/week for 3 months. Behavioral analysis included the Morris Water Maze (MWM). Histological analysis was used to assess the propagation of tau pathology. Results: Animals exposed to EE performed better in the MWM (spatial acquisition duration and total distance, probe test); unexposed animals improved over the course of acquisition trials, but their mean performance remained below that of the EE group. Enriched environment abrogated tau propagation and hippocampal tangle formation in the 600 ng group; in the 900 ng group, tangle formation was ∼10-fold of the 600 ng group, and unaffected by EE. Conclusion: Even a small difference in the amount of injected human AD tau can cause a pronounced difference in the number of resulting tangles. EE leads to a noticeably better spatial navigation performance of tau-injected animals. Furthermore, EE seems to be able to slow down tau pathology progression, indicating the possible utility of similar interventions in early stages of AD where tangle loads are still low.

Synthesis and Assessment of Novel Probes for Imaging Tau Pathology in Transgenic Mouse and Rat Models.

Aggregated tau protein is a core pathology present in several neurodegenerative diseases. Therefore, the development and application of positron emission tomography (PET) imaging radiotracers that selectively bind to aggregated tau in fibril form is of importance in furthering the understanding of these disorders. While radiotracers used in human PET studies offer invaluable insight, radiotracers that are also capable of visualizing tau fibrils in animal models are important tools for translational research into these diseases. Herein, we report the synthesis and characterization of a novel library of compounds based on the phenyl/pyridinylbutadienylbenzothiazoles/benzothiazolium (PBB3) backbone developed for this application. From this library, we selected the compound LM229, which binds to recombinant tau fibrils with high affinity (Kd = 3.6 nM) and detects with high specificity (a) pathological 4R tau aggregates in living cultured neurons and mouse brain sections from transgenic human P301S tau mice, (b) truncated human 151-351 3R (SHR24) and 4R (SHR72) tau aggregates in transgenic rat brain sections, and (c) tau neurofibrillary tangles in brain sections from Alzheimer's disease (3R/4R tau) and progressive supranuclear palsy (4R tau). With LM229 also shown to cross the blood-brain barrier in vivo and its effective radiolabeling with the radioisotope carbon-11, we have established a novel platform for PET translational studies using rodent transgenic tau models.

Corrigendum: Genetic Background Influences the Propagation of Tau Pathology in Transgenic Rodent Models of Tauopathy.

[This corrects the article DOI: 10.3389/fnagi.2019.00343.].

Novel Diagnostic Tools for Identifying Cognitive Impairment in Dogs: Behavior, Biomarkers, and Pathology.

Canine cognitive dysfunction syndrome (CCDS) is a progressive neurodegenerative disorder in senior dogs that is mainly associated with decreased ability to learn and respond to stimuli. It is commonly under-diagnosed because behavioral changes are often attributed to the natural process of aging. In the present study, we used for the first time a comprehensive approach enabling early diagnosis of canine patients with mild cognitive disorders (MiCI). We included CAnine DEmentia Scale (CADES) questionnaires, biochemical parameters, and biomarkers in blood serum, and correlated them with post-mortem histopathological changes. The CADES questionnaires enabled us to identify MiCI dogs developing changes mainly in domains corresponding to social interaction and spatial orientation, which seems to be crucial for delineating early cognitive disorders. Biochemical analyses in these dogs showed slightly elevated liver enzyme parameters (AST and ALT) and significantly decreased sodium and chloride levels in blood serum. Furthermore, we describe for the first time a significant increase of neurofilament light chain (NFL) in blood serum of MiCI dogs, compared to normal aging seniors and young controls, but no changes in TAU protein and amyloid-ß (Aß42) peptide levels. In canine brains with cognitive impairment, amyloid plaques of mainly diffuse and dense types were detected. Furthermore, activated microglia with amoeboid body and dystrophic processes occurred, in some cases with spheroidal and bulbous swellings. On the other hand, no TAU pathology or neurofibrillary tangles were detected. These results suggest that a combination of CADES questionnaire mainly with CNS injury biomarker (NFL) and with biochemical parameters (ALT, AST, Na, and Cl) in blood serum may predict CCDS in senior dogs.

Propagation of Tau Pathology: Integrating Insights From Postmortem and In Vivo Studies.

Cellular accumulation of aggregated forms of the protein tau is a defining feature of so-called tauopathies such as Alzheimer's disease, progressive supranuclear palsy, and chronic traumatic encephalopathy. A growing body of literature suggests that conformational characteristics of tau filaments, along with regional vulnerability to tau pathology, account for the distinct histopathological morphologies, biochemical composition, and affected cell types seen across these disorders. In this review, we describe and discuss recent evidence from human postmortem and clinical biomarker studies addressing the differential vulnerability of brain areas to tau pathology, its cell-to-cell transmission, and characteristics of the different strains that tau aggregates can adopt. Cellular biosensor assays are increasingly used in human tissue to detect the earliest forms of tau pathology, before overt histopathological lesions (i.e., neurofibrillary tangles) are apparent. Animal models with localized tau expression are used to uncover the mechanisms that influence spreading of tau aggregates. Further, studies of human postmortem-derived tau filaments from different tauopathies injected in rodents have led to striking findings that recapitulate neuropathology-based staging of tau. Furthermore, the recent advent of tau positron emission tomography and novel fluid-based biomarkers render it possible to study the temporal progression of tau pathology in vivo. Ultimately, evidence from these approaches must be integrated to better understand the onset and progression of tau pathology across tauopathies. This will lead to improved methods for the detection and monitoring of disease progression and, hopefully, to the development and refinement of tau-based therapeutics.

First-in-Rat Study of Human Alzheimer's Disease Tau Propagation.

One of the key features of misfolded tau in human neurodegenerative disorders is its propagation from one brain area into many others. In the last decade, in vivo tau spreading has been replicated in several mouse transgenic models expressing mutated human tau as well as in normal non-transgenic mice. In this study, we demonstrate for the first time that insoluble tau isolated from human AD brain induces full-blown neurofibrillary pathology in a sporadic rat model of tauopathy expressing non-mutated truncated tau protein. By using specific monoclonal antibodies, we were able to monitor the spreading of tau isolated from human brain directly in the rat hippocampus. We found that exogenous human AD tau was able to spread from the area of injection and induce tau pathology. Interestingly, solubilisation of insoluble AD tau completely abolished the capability of tau protein to induce and spread of neurofibrillary pathology in the rat brain. Our results show that exogenous tau is able to induce and drive neurofibrillary pathology in rat model for human tauopathy in a similar way as it was described in various mouse transgenic models. Rat tau spreading model has many advantages over mouse and other organisms including size and complexity, and thus is highly suitable for identification of pathogenic mechanism of tau spreading.

Genetic Background Influences the Propagation of Tau Pathology in Transgenic Rodent Models of Tauopathy.

Alzheimer's disease (AD), the most common tauopathy, is an age-dependent, progressive neurodegenerative disease. Epidemiological studies implicate the role of genetic background in the onset and progression of AD. Despite mutations in familial AD, several risk factors have been implicated in sporadic AD, of which the onset is unknown. In AD, there is a sequential and hierarchical spread of tau pathology to other brain areas. Studies have strived to understand the factors that influence this characteristic spread. Using transgenic rat models with different genetic backgrounds, we reported that the genetic background may influence the manifestation of neurofibrillary pathology. In this study we investigated whether genetic background has an influence in the spread of tau pathology, using hippocampal inoculations of insoluble tau from AD brains in rodent models of tauopathy with either a spontaneously hypertensive (SHR72) or Wistar-Kyoto (WKY72) genetic background. We observed that insoluble tau from human AD induced AT8-positive neurofibrillary structures in the hippocampus of both lines. However, there was no significant difference in the amount of neurofibrillary structures, but the extent of spread was prominent in the W72 line. On the other hand, we observed significantly higher levels of AT8-positive structures in the parietal and frontal cortical areas in W72 when compared to SHR72. Interestingly, we also observed that the microglia in these brain areas in W72 were predominantly phagocytic in morphology (62.4% in parietal and 47.3% in frontal), while in SHR72 the microglia were either reactive or ramified (67.2% in parietal and 84.7% in frontal). The microglia in the hippocampus and occipital cortex in both lines were reactive or ramified structures. Factors such as gender or age are not responsible for the differences observed in these animals. Put together, our results, for the first time, show that the immune response modulating genetic variability is one of the factors that influences the propagation of tau neurofibrillary pathology.