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1.
Molecules ; 27(7)2022 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-35408484

RESUMO

Cell culturing methods in its classical 2D approach have limitations associated with altered cell morphology, gene expression patterns, migration, cell cycle and proliferation. Moreover, high throughput drug screening is mainly performed on 2D cell cultures which are physiologically far from proper cell functions resulting in inadequate hit-compounds which subsequently fail. A shift to 3D culturing protocols could solve issues with altered cell biochemistry and signaling which would lead to a proper recapitulation of physiological conditions in test systems. Here, we examined porous ultra-high molecular weight polyethylene (UHMWPE) as an inexpensive and robust material with varying pore sizes for cell culturing. We tested and developed culturing protocols for immortalized human neuroblastoma and primary mice hippocampal cells which resulted in high rate of cell penetration within one week of cultivation. UHMWPE was additionally functionalized with gelatin, poly-L-lysine, BSA and chitosan, resulting in increased cell penetrations of the material. We have also successfully traced GFP-tagged cells which were grown on a UHMWPE sample after one week from implantation into mice brain. Our findings highlight the importance of UHMWPE use as a 3D matrix and show new possibilities arising from the use of cheap and chemically homogeneous material for studying various types of cell-surface interactions further improving cell adhesion, viability and biocompatibility.


Assuntos
Técnicas de Cultura de Células , Polietilenos , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Camundongos , Peso Molecular , Polietileno/química , Polietilenos/química , Porosidade
2.
Molecules ; 26(23)2021 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-34885933

RESUMO

The accumulation of the various products of alpha-synuclein aggregation has been associated with the etiology and pathogenesis of several neurodegenerative conditions, including both familial and sporadic forms of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). It is now well established that the aggregation and spread of alpha-synuclein aggregation pathology activate numerous pathogenic mechanisms that contribute to neurodegeneration and, ultimately, to disease progression. Therefore, the development of a safe and effective disease-modifying therapy that limits or prevents the accumulation of the toxic intermediate products of alpha-synuclein aggregation and the spread of alpha-synuclein aggregation pathology could provide significant positive clinical outcomes in PD/DLB cohorts. It has been suggested that this goal can be achieved by reducing the intracellular and/or extracellular levels of monomeric and already aggregated alpha-synuclein. The principal aim of this review is to critically evaluate the potential of therapeutic strategies that target the post-transcriptional steps of alpha-synuclein production and immunotherapy-based approaches to alpha-synuclein degradation in PD/DLB patients. Strategies aimed at the downregulation of alpha-synuclein production are at an early preclinical stage of drug development and, although they have shown promise in animal models of alpha-synuclein aggregation, many limitations need to be resolved before in-human clinical trials can be seriously considered. In contrast, many strategies aimed at the degradation of alpha-synuclein using immunotherapeutic approaches are at a more advanced stage of development, with some in-human Phase II clinical trials currently in progress. Translational barriers for both strategies include the limitations of alpha-synuclein aggregation models, poor understanding of the therapeutic window for the alpha-synuclein knockdown, and variability in alpha-synuclein pathology across patient cohorts. Overcoming such barriers should be the main focus of further studies. However, it is already clear that these strategies do have the potential to achieve a disease-modifying effect in PD and DLB.


Assuntos
Sinucleinopatias/terapia , Animais , Gerenciamento Clínico , Sistemas de Liberação de Medicamentos , Terapia Genética , Humanos , Imunoterapia , Agregados Proteicos/efeitos dos fármacos , Sinucleinopatias/genética , Sinucleinopatias/metabolismo , Sinucleinopatias/patologia , alfa-Sinucleína/análise , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
3.
Transgenic Res ; 30(6): 867-873, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34590209

RESUMO

Conditional pan-neuronal inactivation of the Snca gene in 2-month old male and female mice causes dramatic decrease in the level of the encoded protein, alpha-synuclein, in three studied brain regions, namely cerebral cortex, midbrain and striatum, 12 weeks after the last injection of tamoxifen. Kinetics of alpha-synuclein depletion is different in these brain regions with a longer lag period in the cerebral cortex where this protein is normally most abundant. Our results suggest that efficient post-developmental pan-neuronal knockout of alpha-synuclein in adult, i.e. 5- to 6-month old, animals, could be achieved by tamoxifen treatment of 2-month old mice carrying loxP-flanked Snca gene and expressing inducible Cre-ERT2 recombinase under control of the promoter of neuron-specific enolase (NSE) gene.


Assuntos
Tamoxifeno , alfa-Sinucleína , Animais , Encéfalo/metabolismo , Feminino , Integrases/genética , Integrases/metabolismo , Cinética , Masculino , Camundongos , Camundongos Transgênicos , Recombinação Genética , Tamoxifeno/farmacologia , alfa-Sinucleína/genética
4.
Mol Brain ; 13(1): 75, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393371

RESUMO

Previous studies of the alpha-synuclein null mutant mice on the C57Bl6 genetic background have revealed reduced number of dopaminergic neurons in their substantia nigra pars compacta (SNpc). However, the presence in genomes of the studied mouse lines of additional genetic modifications that affect expression of genes located in a close proximity to the alpha-synuclein-encoding Snca gene makes these data open to various interpretations. To unambiguously demonstrate that the absence of alpha-synuclein is the primary cause of the observed deficit of dopaminergic neurons, we employed a recently produced constituent alpha-synuclein knockout mouse line B6(Cg)-Sncatm1.2Vlb/J. The only modification introduced to the genome of these mice is a substitution of the first coding exon and adjusted short intronic fragments of the Snca gene by a single loxP site. We compared the number of dopaminergic neurons in the SNpc of this line, previously studied B6(Cg)-Sncatm1Rosl/J line and wild type littermate mice. A similar decrease was observed in both knockout lines when compared with wild type mice. In a recently published study we revealed no loss of dopaminergic neurons following conditional inactivation of the Snca gene in neurons of adult mice. Taken together, these results strongly suggest that alpha-synuclein is required for efficient survival or maturation of dopaminergic neurons in the developing SNpc but is dispensable for survival of mature SNpc dopaminergic neurons.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Parte Compacta da Substância Negra/metabolismo , Substância Negra/metabolismo , alfa-Sinucleína/metabolismo , Animais , Sobrevivência Celular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Parte Compacta da Substância Negra/crescimento & desenvolvimento , Substância Negra/citologia , Substância Negra/crescimento & desenvolvimento , alfa-Sinucleína/genética
5.
Neurobiol Aging ; 91: 76-87, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32224067

RESUMO

The etiology and pathogenesis of Parkinson's disease (PD) are tightly linked to the gain-of-function of α-synuclein. However, gradual accumulation of α-synuclein aggregates in dopaminergic neurons of substantia nigra pars compacta (SNpc) leads to the depletion of the functional pool of soluble α-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 α-synuclein, and here we used its tamoxifen-inducible pan-neuronal inactivation to study consequences of the adult-onset (from the age of 6 months) and late-onset (from the age of 12 months) α-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 α-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 α-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.


Assuntos
Envelhecimento/genética , Envelhecimento/patologia , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Técnicas de Inativação de Genes , Doença de Parkinson/etiologia , Doença de Parkinson/genética , Substância Negra/metabolismo , Substância Negra/patologia , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Família Aldeído Desidrogenase 1/genética , Família Aldeído Desidrogenase 1/metabolismo , Animais , Modelos Animais de Doenças , Dopamina/metabolismo , Regulação para Baixo , Expressão Gênica/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terapia de Alvo Molecular , Doença de Parkinson/terapia , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismo , Transmissão Sináptica/genética
6.
Neurochem Res ; 45(5): 1168-1179, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32157564

RESUMO

A number of mutations in a gene encoding RNA-binding protein FUS have been linked to the development of a familial form of amyotrophic lateral sclerosis known as FUS-ALS. C-terminal truncations of FUS by either nonsense or frameshift mutations lead to the development of FUS-ALS with a particularly early onset and fast progression. However, even in patients bearing these highly pathogenic mutations the function of motor neurons is not noticeably compromised for at least a couple of decades, suggesting that until cytoplasmic levels of FUS lacking its C-terminal nuclear localisation signal reaches a critical threshold, motor neurons are able to tolerate its permanent production. In order to identify how the nervous system responds to low levels of pathogenic variants of FUS we produced and characterised a mouse line, L-FUS[1-359], with a low neuronal expression level of a highly aggregation-prone and pathogenic form of C-terminally truncated FUS. In contrast to mice that express substantially higher level of the same FUS variant and develop severe early onset motor neuron pathology, L-FUS[1-359] mice do not develop any clinical or histopathological signs of motor neuron deficiency even at old age. Nevertheless, we detected substantial changes in the spinal cord transcriptome of these mice compared to their wild type littermates. We suggest that at least some of these changes reflect activation of cellular mechanisms compensating for the potentially damaging effect of pathogenic FUS production. Further studies of these mechanism might reveal effective targets for therapy of FUS-ALS and possibly, other forms of ALS.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Doenças Assintomáticas , Perfilação da Expressão Gênica/métodos , Proteína FUS de Ligação a RNA/biossíntese , Medula Espinal/metabolismo , Transcriptoma/fisiologia , Esclerose Amiotrófica Lateral/genética , Animais , Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Proteína FUS de Ligação a RNA/genética
7.
Genes Brain Behav ; 18(8): e12607, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31437340

RESUMO

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 substantially lower level of transgene expression in motor neurons. In a set of tests 5-month old mice displayed certain behavioural abnormalities, including increased impulsivity, decreased anxiety and compromised social interaction, which recapitulate behaviour characteristics typically seen in FTLD patients.


Assuntos
Comportamento Animal , Demência Frontotemporal/genética , Proteína FUS de Ligação a RNA/genética , Animais , Condicionamento Clássico , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/metabolismo , Movimento , Comportamento Social , Transgenes
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