Tomas Smolek

Tau hyperphosphorylation in synaptosomes and neuroinflammation are associated with canine cognitive impairment

Canine cognitive impairment syndrome (CDS) represents a group of symptoms related to the aging of the canine brain. These changes ultimately lead to a decline of memory function and learning abilities, alteration of social interaction, impairment of normal housetraining, and changes in sleep–wake cycle and general activity. We have clinically examined 215 dogs, 28 of which underwent autopsy. With canine brains, we performed extensive analysis of pathological abnormalities characteristic of human Alzheimers disease and frontotemporal lobar degeneration, including β‐amyloid senile plaques, tau neurofibrillary tangles, and fused in sarcoma (FUS) and TAR DNA‐binding protein 43 (TDP43) inclusions. Most demented dogs displayed senile plaques, mainly in the frontal and temporal cortex. Tau neurofibrillary inclusions were found in only one dog. They were identified with antibodies used to detect tau neurofibrillary lesions in the human brain. The inclusions were also positive for Gallyas silver staining. As in humans, they were distributed mainly in the entorhinal cortex, hippocampus, and temporal cortex. On the other hand, FUS and TDP43 aggregates were not present in any of the examined brain samples. We also found that CDS was characterized by the presence of reactive and senescent microglial cells in the frontal cortex. Our transcriptomic study revealed a significant dysregulation of genes involved in neuroinflammation. Finally, we analyzed tau phosphoproteome in the synaptosomes. Proteomic studies revealed a significant increase of hyperphosphorylated tau in synaptosomes of demented dogs compared with nondemented dogs. This study suggests that cognitive decline in dogs is related to the tau synaptic impairment and neuroinflammation. J. Comp. Neurol. 524:874–895, 2016.

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 Alzheimers 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.

Human Truncated Tau Induces Mature Neurofibrillary Pathology in a Mouse Model of Human Tauopathy

Alzheimers disease (AD) represents the most common neurodegenerative disorder. Several animal models have been developed in order to test pathophysiological mechanisms of the disease and to predict effects of pharmacological interventions. Here we examine the molecular and behavioral features of R3m/4 transgenic mice expressing human non-mutated truncated tau protein (3R tau, aa151-391) that were previously used for efficacy testing of passive tau vaccine. The mouse model reliably recapitulated crucial histopathological features of human AD, such as pre-tangles, neurofibrillary tangles, and neuropil threads. The pathology was predominantly located in the brain stem. Transgenic mice developed mature sarkosyl insoluble tau complexes consisting of mouse endogenous and human truncated and hyperphosphorylated forms of tau protein. The histopathological and biochemical features were accompanied by significant sensorimotor impairment and reduced lifespan. The sensorimotor impairment was monitored by a highly sensitive, fully-automated tool that allowed us to assess early deficit in gait and locomotion. We suggest that the novel transgenic mouse model can serve as a valuable tool for analysis of the therapeutic efficacy of tau vaccines for AD therapy.

Transmission of Tau Pathology from Human to Rodent Brain: How to Humanise Animal Models for Alzheimer’s Disease Research

Tauopathies represent a group of neurodegenerative disorders characterised by the accumulation of conformationally altered tau protein. Alzheimer’s disease (AD) is the most prevalent primary tauopathy. In AD, tau pathology progressively spreads across a stereotypical sequence of anatomically connected brain regions. In early stages, the disease manifests in the locus coeruleus and entorhinal cortex; at later stages it spreads through the hippocampus to cortical brain areas. Recent studies suggest that spreading of pathological tau occurs predominantly through neuron-to-neuron transmission; however, glial cells can also be involved in this process. Propagation depends on the conformational state and post-translational modifications of tau protein of various tau strains. Abnormal tau can subsequently act as a seed, misfolding and aggregating normal tau proteins inside the cells. Several research groups have successfully recapitulated tau transmission in animal models. Currently, we are able to induce and drive tau neurodegeneration by using tau species isolated from diseased human brains. Such state-of-the-art “humanised” animal models represent a powerful tool for development of new drug leads and diagnostics for human tauopathies.

Neuronal Expression of Truncated Tau Efficiently Promotes Neurodegeneration in Animal Models: Pitfalls of Toxic Oligomer Analysis

Animal models of neurodegeneration induced by neuronal expression of truncated tau protein emerge as an important tool for understanding the pathogenesis of human tauopathies and for therapy development. Here we highlight common features of truncated tau models and make a critical assessment of possible pitfalls in their analysis. Particularly, the amount of soluble tau oligomers, which are suspected to be neurotoxic agents participating on the spreading of pathology inside the brain, may be overestimated due to a post-lysis oxidative tau oligomerization. Using a mouse brain lysate spiked with recombinant truncated and full length tau forms, we show that tau oligomers might inadvertently be produced during the isolation procedure. This finding is further corroborated by the analysis of brain lysates originated from a mouse model expressing truncated tau variant. Our results underline the necessity of thiol-protecting conditions during the analysis of tau oligomers involved in the etiopathogenesis of various tauopathies including Alzheimers disease.

A comparative study on pathological features of transgenic rat lines expressing either three or four repeat misfolded tau

Human tauopathies represent a heterogeneous group of neurodegenerative disorders characterized by distinct clinical features, typical histopathological structures, and defined ratio(s) of three‐repeat and four‐repeat tau isoforms within pathological aggregates. How the optional microtubule‐binding repeat of tau influences this differentiation of pathologies is understudied. We have previously generated and characterized transgenic rodent models expressing human truncated tau aa151–391 with either three (SHR24) or four microtubule‐binding repeats (SHR72). Here, we compare the behavioral and neuropathological hallmarks of these two transgenic lines using a battery of tests for sensorimotor, cognitive, and neurological functions over the age range of 3.5–15 months. Progression of sensorimotor and neurological deficits was similar in both transgenic lines; however, the lifespan of transgenic line SHR72 expressing truncated four‐repeat tau was markedly shorter than SHR24. Moreover, the expression of three or four‐repeat tau induced distinct neurofibrillary pathology in these lines. Transgenic lines displayed different distribution of tau pathology and different type of neurofibrillary tangles. Our results suggest that three‐ and four‐repeat isoforms of tau may display different modes of action in the diseased brain.

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.

Correction: Cizkova, D., et al. Localized Intrathecal Delivery of Mesenchymal Stromal Cells Conditioned Media Improves Functional Recovery in A Rat Model of Contusive Spinal Cord Injury. Int. J. Mol. Sci. 2018, 19, 870

Dasa Cizkova 1,2,3,*, Veronika Cubinkova 1, Tomas Smolek 1, Adriana-Natalia Murgoci 1,2,3, Jan Danko 2, Katarina Vdoviakova 2, Filip Humenik 2, Milan Cizek 4, Jusal Quanico 3 ID , Isabelle Fournier 3 and Michel Salzet 3 ID 1 Institute of Neuroimmunology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 10 Bratislava, Slovakia; veronica.cubinkova@savba.sk (V.C.); tomas.smolek@savba.sk (T.S.); adriana.murgoci@savba.sk (A.-N.M.) 2 Department of Anatomy, Histology and Physiology, University of Veterinary Medicine and Pharmacy in Košice, Komenského 73, 041 81 Košice, Slovakia; jan.danko@uvlf.sk (J.D.); katarina.vdoviakova@uvlf.sk (K.V.); filip.humenik@uvlf.sk (F.H.) 3 University Lille, Inserm, U-1192—Laboratoire Protéomique, Réponse Inflammatoire et Spectrométrie de Masse-PRISM, F-59000 Lille, France; Jusal.Quanico@univ-lille1.fr (J.Q.); isabelle.fournier@univ-lille1.fr (I.F.); michel.salzet@univ-lille1.fr (M.S.) 4 Department of Epizootology and Parasitology, University of Veterinary Medicine and Pharmacy in Košice, Komenského 73, 041 81 Košice, Slovakia; milan.cizek@uvlf.sk * Correspondence: dasa.cizkova@uvlf.sk; Tel.: +421-2-5478-8100; Fax: +421-2-5477-4276