Papers by Michail Kukharsky
CNS Neuroscience & Therapeutics
AimsTo assess effects of DF402, a bioisostere of Dimebon/Latrepirdine, on the disease progression... more AimsTo assess effects of DF402, a bioisostere of Dimebon/Latrepirdine, on the disease progression in the transgenic model of amyotrophic lateral sclerosis (ALS) caused by expression of pathogenic truncated form of human FUS protein.MethodsMice received DF402 from the age of 42 days and the onset of clinical signs, the disease duration and animal lifespan were monitored for experimental and control animals, and multiple parameters of their gait were assessed throughout the pre‐symptomatic stage using CatWalk system followed by a bioinformatic analysis. RNA‐seq was used to compare the spinal cord transcriptomes of wild‐type, untreated, and DF402‐treated FUS transgenic mice.ResultsDF402 delays the onset and slows the progression of pathology. We developed a CatWalk analysis protocol that allows detection of gait changes in FUS transgenic mice and the effect of DF402 on their gait already at early pre‐symptomatic stage. At this stage, a limited number of genes significantly change expre...
Figure S6. Dose-dependent toxicity of enoxacin, edaravone and riluzole. a SH-SY5Y cells were trea... more Figure S6. Dose-dependent toxicity of enoxacin, edaravone and riluzole. a SH-SY5Y cells were treated with corresponding doses of compounds for 24 h, and toxicity was assessed using CellTiter Blue® Cell Viability Assay. *p
Figure S5. Differentiation of human neuroblastoma cells into neuron-like cells does not lead to t... more Figure S5. Differentiation of human neuroblastoma cells into neuron-like cells does not lead to the loss of paraspeckles. Differentiated SH-SY5Y cells develop extensive neurite network and are uniformly positive for a neuronal marker Tuj 1 (left panel) but preserve their ability to form paraspeckles (right panel). SH-SY5Y cells were induced to differentiate into neuron-like cells using retinoic acid/BDNF and analysed 6 days into differentiation by immunocytochemitry and NEAT1_2 RNA-FISH. Representative images are shown. Scale bars, 100 μm (left panel) and 10 μm (right panel). (DOCX 175 kb)
Figure S4. The effect of IFN-inducing ligands on NEAT1 and paraspeckles. a IFNbeta is robustly in... more Figure S4. The effect of IFN-inducing ligands on NEAT1 and paraspeckles. a IFNbeta is robustly induced by poly(I:C) in neuroblastoma cells. Cells were analysed by qRT-PCR after 24 h of poly(I:C) stimulation (n = 5). **p
Figure S3. Accumulation of dsRNA (a) and increased levels of p-eIF2α (b) in MCF7 cells depleted o... more Figure S3. Accumulation of dsRNA (a) and increased levels of p-eIF2α (b) in MCF7 cells depleted of TDP-43. Cells were analysed 48 h post-transfection. Scale bars, 100 μm and 10 μm for general plane and close-up panels respectively. (DOCX 663 kb)
Figure S2. Drosha or TDP-43 downregulation affects miRNA processing. a Drosha knockdown leads to ... more Figure S2. Drosha or TDP-43 downregulation affects miRNA processing. a Drosha knockdown leads to accumulation of miRNA precursors, pri-miR-17-92a and pri-miR-15a (n = 4–6). **p
Figure S1. The effect of TDP-43 dysfunction on paraspeckles, speckles and paraspeckle proteins in... more Figure S1. The effect of TDP-43 dysfunction on paraspeckles, speckles and paraspeckle proteins in MCF7 and SH-SY5Y cells. a and b TDP-43 siRNA-mediated knockdown upregulates NEAT1_2 (a) and enhances paraspeckle assembly (b) in SH-SY5Y cells. Cells were transfected with scrambled siRNA or Silencer® TDP-43 siRNA and analysed by qRT-PCR (n = 6). Mean number of paraspeckles per cell was also quantified using NEAT1_2 RNA FISH. **p
Modern technologies in ophtalmology, 2021
Dopaminergic system takes part in regulation of intraocular pressure (IOP) and other elements of ... more Dopaminergic system takes part in regulation of intraocular pressure (IOP) and other elements of the pathogenesis of glaucoma. Development of new drugs for the glaucoma treatment remains an actual problem in modern ophthalmology. Purpose. To estimate the perspective of topical use of dopaminergic system regulators as ocular hypotensive agents. Matherials and methods. The investigation was carried out on healthy Shinshilla rabbits and knock-out mice with γ-sinuclein gene deletion (γ-KO) that served for a model of ocular hypertension. IOP was measured before 10% dopamine instillation and every 30 minutes after it for 2 hours totally. Results. Single instillation of 10% dopamine caused a mean IOP decrease of 4-5 mm Hg after 1,5-2 hours. In γ-KO mice IOP decreased significantly on 5-6 mm Hg after 30 min and remained lower than the initial level for 2 hours. Conclusion. Single instillation of 10% dopamine cause a statistically significant decrease of IOP in healthy rabbits and γ-KO mice ...
Молекулярная биология, 2021
Aggregated forms of α-synuclein are core components of pathohistological inclusions known as Lewy... more Aggregated forms of α-synuclein are core components of pathohistological inclusions known as Lewy bodies in substantia nigra (SN) neurons of patients with Parkinson's disease (PD). The role of α-synuclein in selective loss of SN dopaminergic neurons (DNs) in PD is studied in mice knocked out in the α-synuclein gene. The new mouse strain delta flox KO with a constitutive knockout of the α-synuclein gene models the end point of in vivo deletion of the α-synuclein gene in mice with a conditional knockout and has no foreign sequence in the modified genomic locus, thus differing from all other α-synuclein knockout mouse strains. The effect of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is used to model PD, was compared between delta flox KO mice and mice of the well-known α-synuclein knockout strain AbKO. Subchronic MPTP administration, which models early PD, was found to reduce the dopamine content and to change the ratio of dopamine metabolites in the striatum to the same levels in delta flox KO, АbKO, and wild-type mice. Overt locomotor defects were not observed after MPTP treatment, but gait testing in a CatWalk XT (Noldus) system revealed identical gait deviations in mice of the two strains and control wild-type mice. Based on the findings, a similar mechanism of neurotoxic damage to DNs was assumed for delta flox KO and AbKO mice.
Medicinal Research Reviews, 2020
Recent progress in understanding the pathological changes in the nervous system and in certain ot... more Recent progress in understanding the pathological changes in the nervous system and in certain other body systems (e.g., immune system) that lead to the development and progression of amyotrophic lateral sclerosis (ALS) revealed a number of molecular and cellular processes that can potentially be used as therapeutic targets. Many of these processes are compromised not only in ALS but also in other diseases and a repertoire of drugs able to restore, at least partially, their functionality has been developed. In this review, we briefly describe current approaches to the repurposing of such “old” drugs for treatment of patients with ALS.
Neurobiology of Aging, 2020
The etiology and pathogenesis of Parkinson's disease (PD) are tightly linked to the gain-of-funct... more The etiology and pathogenesis of Parkinson's disease (PD) are tightly linked to the gain-of-function of asynuclein. However, gradual accumulation of a-synuclein aggregates in dopaminergic neurons of substantia nigra pars compacta (SNpc) leads to the depletion of the functional pool of soluble a-synuclein, and therefore, creates loss-of-function conditions, particularly in presynaptic terminals of these neurons. Studies of how this late-onset depletion of a protein involved in many important steps of neurotransmission contributes to PD progression and particularly, to worsening the nigrostriatal pathology at late stages of the disease are limited and obtained data, are controversial. Recently, we produced a mouse line for conditional knockout of the gene encoding a-synuclein, and here we used its tamoxifen-inducible panneuronal inactivation to study consequences of the adult-onset (from the age of 6 months) and late-onset (from the age of 12 months) a-synuclein depletion to the nigrostriatal system. No significant changes of animal balance/coordination, the number of dopaminergic neurons in the SNpc and the content of dopamine and its metabolites in the striatum were observed after adult-onset a-synuclein depletion, but in aging (18-month-old) late-onset depleted mice we found a significant reduction of major dopamine metabolites without changes to the content of dopamine itself. Our data suggest that this might be caused, at least partially, by reduced expression of aldehyde dehydrogenase ALDH1a1 and could lead to the accumulation of toxic intermediates of dopamine catabolism. By extrapolating our findings to a potential clinical situation, we suggest that therapeutic downregulation of a-synuclein expression in PD patients is a generally safe option as it should not cause adverse side effects on the functionality of their nigrostriatal system. However, if started in aged patients, this type of therapy might trigger slight functional changes of the nigrostriatal system with potentially unwanted additive effect to already existing pathology.
Lesion of the dopaminergic neurons of the nigrostriatal system is a key feature of Parkinson'... more Lesion of the dopaminergic neurons of the nigrostriatal system is a key feature of Parkinson's disease (PD). Alpha-synuclein is a protein that is a major component of Lewy bodies, histopathological hallmarks of PD, and is involved in regulation of dopamine (DA) neurotransmission. Previous studies of knockout mice have shown that inactivation of alpha-synuclein gene can lead to the reduction in number of DA neurons in the substantia nigra (SN). DA neurons of the SN are known to be the most affected in PD patients whereas DA neurons of neighboring ventral tegmental area (VTA) are much less susceptible to degeneration. Here we have studied the dynamics of changes in TH-positive cell numbers in the SN and VTA during a critical period of their embryonic development in alpha-synuclein knockout mice. This precise study of DA neurons during development of the SN revealed that not only is the number of DA neurons reduced by the end of the period of ontogenic selection, but that the way t...
NEAT1 is a highly and ubiquitously expressed long non-coding RNA (lncRNA) which serves as an impo... more NEAT1 is a highly and ubiquitously expressed long non-coding RNA (lncRNA) which serves as an important regulator of cellular stress response. However, the physiological role of NEAT1 in the central nervous system (CNS) is still poorly understood. In the current study, we addressed this by characterising the CNS function in the Neat1 knockout mouse model (Neat1-/- mice), using a combination of behavioural phenotyping, electrophysiology and expression analysis. RNAscope® in situ hybridisation revealed that in wild-type mice, Neat1 is expressed evenly across the CNS, with high expression in glial cells and low expression in neurons. Loss of Neat1 in mice results in an inadequate reaction to physiological stress manifested as hyperlocomotion and panic escape response. In addition, Neat1-/- mice display deficits in social interaction and rhythmic patterns of activity but retain normal motor function and memory. Neat1-/- mice do not present with neuronal loss, overt neuroinflammation or g...
Genes, Brain and Behavior, 2019
Multiple clinical and experimental evidences suggest that amyotrophic lateral sclerosis (ALS) and... more Multiple clinical and experimental evidences suggest that amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are members of a disease continuum. Pathological inclusions of fused in sarcoma (FUS) protein have been observed in subsets of patients with these diseases but their anatomical distribution is different for two diseases. These structures are present in motor neurons in ALS cases but in cortical neurons in FTLD cases. Expression of a C‐terminally truncated form of human FUS causes an early onset and progressive motor neuron pathology in transgenic mice but only when these neurons express a certain level of this protein. Severe motor dysfunction and early lethality of mice with expression above this level prevent their use for studies of FTLD‐related pathology caused by expression of this form of FUS. In the present study, we used another line of mice expressing the same protein but not developing any signs of motor system dysfunction due to substa...
Mutations in a gene encoding RNA-binding protein FUS was linked to familial forms of amyotrophic ... more Mutations in a gene encoding RNA-binding protein FUS was linked to familial forms of amyotrophic lateral sclerosis (ALS). C-terminal truncations of FUS are associated with aggressive forms of ALS. However, motor neurons are able to tolerate permanent production of pathogenic truncated form of FUS protein until its accumulation in the cytoplasm of neurones does not reach a critical threshold. In order to identify how the nervous system responds to pathogenic variants of FUS we produced and characterised a mouse line, L-FUS[1-359], with a low level of neuronal expression of a highly aggregation prone and pathogenic form of C-terminally truncated FUS. In contrast to mice with substantially higher level of expression of the same FUS variant that develop severe early onset motor neuron pathology, L-FUS[1-359] mice do not develop any sign of pathology even at old age. Nevertheless, we detected substantial changes in the spinal cord transcriptome of these mice comparing to the wild type li...
Human Cell, 2019
Recent studies have demonstrated that breast milk contains a population of cells displaying many ... more Recent studies have demonstrated that breast milk contains a population of cells displaying many of the properties typical of stem cells. This review outlines progress made in this newly emerging field of stem cell biology and provides an analysis of the available data on purification, propagation and differentiation of certain types of progenitor cells from breast milk. The possible fates of breast milk cells, including microchimerism caused by their transmission to the distant organs of the infant, are also discussed. Unique properties of breast milk-derived stem cells, such as their unusually low tumorigenic potential and their negligible ability to form teratomas, are highlighted as obvious advantages for using these cells in regenerative therapy.
Molecular Neurodegeneration, 2018
Background: Paraspeckles are subnuclear bodies assembled on a long non-coding RNA (lncRNA) NEAT1.... more Background: Paraspeckles are subnuclear bodies assembled on a long non-coding RNA (lncRNA) NEAT1. Their enhanced formation in spinal neurons of sporadic amyotrophic lateral sclerosis (ALS) patients has been reported but underlying mechanisms are unknown. The majority of ALS cases are characterized by TDP-43 proteinopathy. In current study we aimed to establish whether and how TDP-43 pathology may augment paraspeckle assembly. Methods: Paraspeckle formation in human samples was analysed by RNA-FISH and laser capture microdissection followed by qRT-PCR. Mechanistic studies were performed in stable cell lines, mouse primary neurons and human embryonic stem cell-derived neurons. Loss and gain of function for TDP-43 and other microRNA pathway factors were modelled by siRNA-mediated knockdown and protein overexpression. Results: We show that de novo paraspeckle assembly in spinal neurons and glial cells is a hallmark of both sporadic and familial ALS with TDP-43 pathology. Mechanistically, loss of TDP-43 but not its cytoplasmic accumulation or aggregation augments paraspeckle assembly in cultured cells. TDP-43 is a component of the microRNA machinery, and recently, paraspeckles have been shown to regulate pri-miRNA processing. Consistently, downregulation of core protein components of the miRNA pathway also promotes paraspeckle assembly. In addition, depletion of these proteins or TDP-43 results in accumulation of endogenous dsRNA and activation of type I interferon response which also stimulates paraspeckle formation. We demonstrate that human or mouse neurons in vitro lack paraspeckles, but a synthetic dsRNA is able to trigger their de novo formation. Finally, paraspeckles are protective in cells with compromised microRNA/dsRNA metabolism, and their assembly can be promoted by a small-molecule microRNA enhancer. Conclusions: Our study establishes possible mechanisms behind paraspeckle hyper-assembly in ALS and suggests their utility as therapeutic targets in ALS and other diseases with abnormal metabolism of microRNA and dsRNA.
Zhurnal nevrologii i psikhiatrii im. S.S. Korsakova, 2015
В обзоре рассматриваются современные гипотезы патогенеза болезни Альцгеймера (БА) и способы фарма... more В обзоре рассматриваются современные гипотезы патогенеза болезни Альцгеймера (БА) и способы фармакологической коррекции. Они представлены в следующих разделах: роль β-амилоида (Аβ) в патогенезе БА, роль тау-белка в патогенезе БА, основные гипотезы патогенеза, связь между Аβ и тау-белком, дисфункция синапсов при БА, нейроиммуная гипотеза, подходы к лечению БА.
Cell death & disease, Jan 11, 2017
Dysregulation of stress granules (SGs) and their resident proteins contributes to pathogenesis of... more Dysregulation of stress granules (SGs) and their resident proteins contributes to pathogenesis of a number of (neuro)degenerative diseases. Phosphorylation of eIF2α is an event integrating different types of cellular stress and it is required for SG assembly. Phosphorylated eIF2α (p-eIF2α) is upregulated in the nervous system in some neurodegenerative conditions. We found that increasing p-eIF2α level by proteasomal inhibition in cultured cells, including mouse and human neurons, before a SG-inducing stress ('stress preconditioning'), limits their ability to maintain SG assembly. This is due to upregulation of PP1 phosphatase regulatory subunits GADD34 and/or CReP in preconditioned cells and early decline of p-eIF2α levels during subsequent acute stress. In two model systems with constitutively upregulated p-eIF2α, mouse embryonic fibroblasts lacking CReP and brain neurons of tau transgenic mice, SG formation was also impaired. Thus, neurons enduring chronic stress or primed...
Neurotoxicity research, Jan 3, 2016
Intracerebral or intraperitoneal injections of brain extracts from the Alzheimer's disease pa... more Intracerebral or intraperitoneal injections of brain extracts from the Alzheimer's disease patients result in the acceleration of cerebral β-amyloidosis in transgenic mice. Earlier, we have found that intravenous injections of synthetic full-length amyloid-β (Aβ) comprising the isomerized Asp7 trigger cerebral β-amyloidosis. In vitro studies have shown that isomerization of Asp7 promotes zinc-induced oligomerization of the Aβ metal-binding domain (Aβ1-16). Here we report that single intracerebral injection of the peptide Aβ1-16 with isomerized Asp7 (isoAβ1-16) but not the injection of Aβ1-16 significantly increases amyloid burden in 5XFAD transgenic mice. Our results provide evidence for a role of isoAβ1-16 as a minimal seeding agent of Aβ aggregation in vivo.
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Papers by Michail Kukharsky