Neurobiology

Cellular stress can influence efficiency of iPSCs generation and their differentiation. However, the role of intracellular cytoprotective factors in these processes is still not well known. Therefore, we investigated the effect of HO-1 (Hmox1) or Nrf2 (Nfe2l2), two major cytoprotective genes. Hmox1-/fibroblasts demonstrated decreased reprogramming efficiency in comparison to Hmox1 1/1 cells. Reversely, pharmacological enhancement of HO-1 resulted in higher number of iPSCs colonies. Importantly, elevated level of both p53 and p53-regulated miR-34a and 14-3-3r was observed in HO-1-deficient fibroblasts whereas downregulation of p53 in these cells markedly increased their reprogramming efficiency. In human fibroblasts HO-1 silencing also induced p53 expression and affected reprogramming outcome. Hmox1 1/1 and Hmox1-/-iPSCs similarly differentiated in vitro to cells originating from three germ layers, however, lower number of contracting cells was observed during this process in HO-1-deficient cells indicating attenuated cardiac differentiation. Importantly, silencing of Hmox1 in murine ESC using CRISPR/Cas-9 editing also impaired their spontaneous cardiac differentiation. Decreased reprogramming efficiency was also observed in Nrf2-lacking fibroblasts. Reversely, sulforaphane, a Nrf2 activator, increased the number of iPSCs colonies. However, both Nfe2l2 1/1 and Nfe2l2-/-iPSCs showed similar pluripotency and differentiation capacity. These results indicate that regulation of HO-1 expression can further optimize generation and cardiac differentiation of iPSCs.

Herein, we used a clinically-relevant model of 10 min cardiac arrest (CA) in Wistar rats. Histological analyses of the ischemic brains of old rats showed significant atrophy of CA 1 sector of hippocampus (Nissl and NeuN stainings) corresponding with increase of glial fibrillary acidic protein expression. The longterm behavioral consequences of above manipulation producing global brain ischemia were assessed in young, middle-aged and old rats, i.e., 3-, 6-and 18-months post-treatment, respectively. In young animals no differences were found in the context-dependent memory in Fear Conditioning test. The most striking behavioral abnormalities were found in middle-aged rats (6 months post-ischemia). Ischemic rats showed hyperactivity and decreased level of anxiety in Open Field and problems with spatial learning and memory in a Novel Object Location test, T-maze and Morris Water Maze. In old animals, a decline of motor and cognitive functions was found not only in ischemic but also in sham/control ones. This study describes consequences of global brain ischemia in aging animals. contributed equally to this article.

The role of adult brain neurogenesis (generating new neurons) in learning and memory appears to be quite firmly established in spite of some criticism and lack of understanding of what the new neurons serve the brain for. Also, the few experiments showing that blocking adult neurogenesis causes learning deficits used irradiation and various drugs known for their side effects and the results obtained vary greatly. We used a novel approach, cyclin D2 knockout mice (D2 KO mice), specifically lacking adult brain neurogenesis to verify its importance in learning and memory. D2 KO mice and their wild-type siblings were tested in several behavioral paradigms, including those in which the role of adult neurogenesis has been postulated. D2 KO mice showed no impairment in sensorimotor tests, with only sensory impairment in an olfaction-dependent task. However, D2 KO mice showed proper procedural learning as well as learning in context (including remote memory), cue, and trace fear conditioning, Morris water maze, novel object recognition test, and in a multifunctional behavioral system-IntelliCages. D2 KO mice also demonstrated correct reversal learning. Our results suggest that adult brain neurogenesis is not obligatory in learning, including the kinds of learning where the role of adult neurogenesis has previously been strongly suggested.

In the central nervous system (CNS) generation of new neurons continues throughout adulthood, when it is limited to the olfactory bulb and hippocampus. The knowledge regarding the function of newly-generated neurons remains limited and is vigorously investigated using diverse approaches. Among these are genetically modified mice, most of them of knock-out type (KO). Results from 23 diverse KO mouse models demonstrate the importance of particular proteins (growth factors, nitric oxide synthases, receptors, cyclins/cyclin-associated proteins, transcription factors, etc.) in adult neurogenesis (ANGE) as well as separate it from developmental neurogenesis. These results bring us closer to revealing the function of newly generated neurons in adult brains.

In the present study, we used a new training paradigm in the intelliCage automatic behavioral assessment system to investigate cognitive functions of the transgenic mice harboring London mutation of the human amyloid precursor protein (APP.V717I). Three groups of animals: 5-, 12-and 18-24-month old were subjected to both Water Maze training and the IntelliCage-based appetitive conditioning. The spatial memory deficit was observed in all three groups of transgenic mice in both behavioral paradigms. However, the APP mice were capable to learn normally when co-housed with the wild-type (WT) littermates, in contrast to clearly impaired learning observed when the transgenic mice were housed alone. Furthermore, in the transgenic mice kept in the Intellicage alone, the cognitive deficit of the young animals was modulated by the circadian rhythm, namely was prominent only during the active phase of the day. The novel approach to study the transgenic mice cognitive abilities presented in this paper offers new insight into cognitive dysfunctions of the Alzheimer's disease mouse model.

 The BRCA1 protein, one of the major players responsible for DNA damage response has recently been linked to Alzheimer's disease (AD). Using primary fibroblasts and neurons reprogrammed from induced pluripotent stem cells (iPSC) derived from familial AD (FAD) patients, we studied the role of the BRCA1 protein underlying molecular neurodegeneration. By whole-transcriptome approach, we have found wide range of disturbances in cell cycle and DNA damage response in FAD fibroblasts. This was manifested by significantly increased content of BRCA1 phosphorylated on Ser1524 and abnormal ubiquitination and subcellular distribution of presenilin 1 (PS1). Accordingly, the iPSC-derived FAD neurons showed increased content of BRCA1(Ser1524) colocalized with degraded PS1, accompanied by an enhanced immunostaining pattern of amyloid-β. Finally, overactivation of BRCA1 was followed by an increased content of Cdc25C phosphorylated on Ser216, likely triggering cell cycle re-entry in FAD neurons. ...

Current Alzheimer's disease (AD) diagnostics is based on cognitive testing, and detecting amyloid Aβ and τ pathology by brain imaging and assays of cerebrospinal fluid. However, biomarkers identifying complex pathways contributing to pathology are lacking, especially for early AD. Preferably, such biomarkers should be more cost-effective and present in easily available diagnostic tissues, such as blood. Here, we summarize the recent findings of potential early AD molecular diagnostic biomarkers in blood platelets, lymphocytes and erythrocytes. We review molecular alterations which refer to such main hypotheses of AD pathogenesis as amyloid cascade, oxidative and mitochondrial stress, inflammation and alterations in cell cycle regulatory molecules. The major advantage of such biomarkers is the potential ability to indicate individualized therapies in AD patients.

In Alzheimer's disease (AD), molecular changes are observed not only in patients' neurons but also in peripheral cells, such as blood lymphocytes. These include changes in the level of oxidative stress markers, mitochondria impairment, and aberrant cell cycle regulation in AD blood lymphocytes. While the concepts of early causes of AD are currently highly controversial, these findings provide support for the cell cycle hypothesis of AD pathomechanism and emphasize the systemic nature of the disease. Moreover, because of difficulties in studying dynamic processes in the human brain, lymphocytes seem to be useful for readout of AD molecular mechanisms. In addition, lymphocytes as easily accessible human cells have potential diagnostic value. We summarize current perspectives for the development of new therapeutic strategies based on oxidative stress and cell cycle dysregulation in AD, and for diagnostic methodologies involving new markers in AD lymphocytes.

Alzheimer's disease (AD) is the most common age-related dementia. Among its major challenges is identifying molecular signatures characteristic for the early AD stage in patients with Mild Cognitive Impairment (MCI-AD), which could serve for deciphering the AD pathomechanism and also as non-invasive, easy-to-access biomarkers. Using qRT-PCR we compared the microRNA (miRNA) profiles in blood plasma of 15 MCI-AD patients, whose diagnoses were confirmed by cerebrospinal fluid (CSF) biomarkers, with 20 AD patients and 15 non-demented, age-matched individuals (CTR).To minimize methodological variability, we adhered to standardization of blood and CSF assays recommended by the international Joint Programming for Neurodegenerative Diseases (JPND) BIOMARKAPD consortium, and we employed commercially available Exiqon qRT-PCR-assays. In the first screening, we assessed 179 miRNAs of plasma. We confirmed 23 miRNAs reported earlier as AD biomarker candidates in blood and found 26 novel diffe...

Familial (FAD) and sporadic (SAD) Alzheimer's disease do not share all pathomechanisms, but knowledge on their molecular differences is limited. We previously reported that cell cycle control distinguishes lymphocytes from SAD and FAD patients. Significant differences were found in p21 levels of SAD compared to FAD lymphocytes. Since p21 can also regulate apoptosis, the aim of this study was to compare the response of FAD and SAD lymphocytes to oxidative stress like 2-deoxy-D-ribose (2dRib) treatment and to investigate the role of p21 levels in this response. We report that FAD cells bearing seven different PS1 mutations are more resistant to 2dRib-induced cell death than control or SAD cells: FAD cells showed a lower apoptosis rate and a lower depolarization of the mitochondrial membrane. Despite that basal p21 cellular content was lower in FAD than in SAD cells, in response to 2dRib, p21 mRNA and protein levels significantly increased in FAD cells. Moreover, we found a higher ...

Alzheimer’s disease (AD) is the most common cause of dementia and a great socioeconomic burden in the aging society. Compelling evidence demonstrates that molecular change characteristics for AD, such as oxidative stress and amyloidβ(Aβ) oligomerization, precede by decades the onset of clinical dementia and that the disease represents a biological and clinical continuum of stages, from asymptomatic to severely impaired. Nevertheless, the sequence of the early molecular alterations and the interplay between them are incompletely understood. This review presents current knowledge about the oxidative stress-induced impairments and compromised oxidative stress defense mechanisms in AD brain and the cross-talk between various pathophysiological insults, with the focus on excessive reactive oxygen species (ROS) generation and Aβoverproduction at the early stages of the disease. Prospects for AD therapies targeting oxidant/antioxidant imbalance are being discussed, as well as for the devel...

Induction of mitotic catastrophe through the disruption of microtubules is an established target in cancer therapy. However, the molecular mechanisms determining the mitotic catastrophe and the following apoptotic or non-apoptotic cell death remain poorly understood. Moreover, many existing drugs targeting tubulin, such as vincristine, have reduced efficacy, resulting from poor solubility in physiological conditions. Here, we introduce a novel small molecule 2-aminoimidazoline derivative—OAT-449, a synthetic water-soluble tubulin inhibitor. OAT-449 in a concentration range from 6 to 30 nM causes cell death of eight different cancer cell lines in vitro, and significantly inhibits tumor development in such xenograft models as HT-29 (colorectal adenocarcinoma) and SK-N-MC (neuroepithelioma) in vivo. Mechanistic studies showed that OAT-449, like vincristine, inhibited tubulin polymerization and induced profound multi-nucleation and mitotic catastrophe in cancer cells. HeLa and HT-29 cel...

Due to wide range of biological actions of sulfonamides, quinoline derivatives and 1,2,3-triazoles, as well as presence of this structures in substances bearing anticancer, antibacterial, antitubercular or beta-lactamase inhibiting activities, an effort was made to find an effective, one-pot method for synthesis of derivatives containing those three moieties.

Five "click chemistry" synthesis methods were evaluated:
-single-step CuAAC in DMFaq (A1) and tert-butanol/water (A2),
-single-step PTC method (B),
-two-step method using organic azide (C),
-single-step catalyst free (D).
The substrates were several 4-amino-3-quinolinesulfonamide analogs containing a propargyl group and four different halide types: benzyl bromide and chloride, allyl bromide and 1-bromo-3-methyl-2-buten.

13 new, not published in literature compounds were obtained. They were derivatives of: 4-amino-(N-methyl-1,2,3-triazolyl)-3-quinolinesulfonamides, 4-amino-3-quinoline-N-(4-methyl-1,2,3-triazolyl)sulfonamides and
4-chloro-3-quinoline-N-(methyl-1,2,3-triazolyl)sulfonamides. Yields were between 31 and 99%. Benzyl halide substrates yielded 65-99%, 1-bromo-3-methyl-2-butenu yielded 83-97%, and allyl bromide yielded 31-58%. There was no clear dependence of reaction yields on activity of halide type which was used as substrate.

It was concluded, that best methods for such syntheses are A1, A2, B and C. In most cases the only product was 1,4-disubstituted triazole. Method D has limited significance. For several obtained products a phenomenon similar to polymorphism may occur.

Introduction
Sulfonamides have many biological activities, i.e. bacteriostatic, diuretic, anti-diabetic or anti-inflammatory. The discovery of an anti-cancer sulfonamide E7010 showed a new potential activity. Molecules containing 1,2,3-triazole group have strong activities too i.e. bactericidal, fungicidal, anti-inflammatory, anti-viral
and anti-neoplastic.

Aim of the study
Aim of the research was to find an effective one-pot, click-chemistry reaction to synthesize quinoline-based compounds with both sulfonamide and triazole groups. Obtained derivatives were to be tested for anti-neoplastic activity.

Material and methods
15 various 4-propargylamino-3-quinolinesulfonamides and 4-amino-3-quinoline-N-propargylsulfonamides were obtained from 4-chloro-3-quinolinesulfonamide chloride. Tested on cancer cell colonies, some of them showed moderate activity. They were used in 5 variants of azide-alkyne 1,3-cycloaddition:
• catalyst-free, solvent: H2O,
• two-phase with phase transfer catalyst (TEBA-Br),
• copper-catalyzed azide-alkyne cycloaddition (CuAAC),
solvents: DMF/H2O or tert-butanol,
• two-step: organic azide sythesis before CuAAC.
The reactions were made with 3 different halides: benzyl, allyl and 3-methyl-2-buten halide. Product structures were determined by 1H NMR and MS.

Results
13 derivatives of 4-amine-(N-4-methyl-1,2,3-triazol-4-yl)-3-quinolinesulfonamides and 4-amine-3-quinoline-N-(4-methyl-1,2,3-triazol-4-yl)sulfonamides were obtained. Benzyl halides yielded 80-90%, 3-methyl-2-buten halides: 70-80%, allyl halides: 40-50%.
Catalyst-free reaction did not occur, due to lack of substrate solubility. Thus it can be applied only for liquids creating water emulsions. Two-phase reaction caused difficulties in product purifying from TEBA-Br remains. Two-step reaction had good yields, but required organic azide synthesis before actual cycloaddition. One-pot CuAAC had good yields and simple ways of product separation: filtration or extraction.

Conclusion
The two last mentioned  methods seem to be most effective and variable in applications. The one-pot CuAAC may be best for chemical and pharmaceutical industry, due to its simplicity and short reaction time (24 to 48h).