Nina Kraskovskaya

Background/Objectives: Patient-derived cell lines retaining donor-specific age-related and genomic features are essential for modeling late-onset neurodegenerative disorders like Huntington’s disease (HD). This study aims to establish and comprehensively characterize HDDF2, a novel dermal fibroblast line from an HD patient, to provide a relevant cellular model. Methods: Dermal fibroblasts were isolated and cultured from a 44-year-old male HD patient carrying 46 CAG repeats in the HTT gene. Cells were evaluated for senescence markers (p16, lamin B1, SA-β-Gal activity, proliferation rates) and polyglutamine (polyQ) aggregation. Direct reprogramming protocols were applied to convert these fibroblasts into induced neurons. Results: HDDF2 fibroblasts exhibited a pronounced senescence-associated phenotype, evidenced by increased p16 expression, reduced lamin B1 levels, elevated SA-β-Gal activity, and decreased proliferation. Notably, polyQ deposition was preferentially detected within the senescent subpopulation, displaying distinct localization patterns differentiating senescent from proliferating cells. Despite this, HDDF2 cells retained their capacity for direct reprogramming and were successfully converted into induced neurons. Conclusions: HDDF2 represents a well-characterized, patient-specific cellular model for HD. The observed co-occurrence of polyQ deposition and cellular senescence, combined with successful neuronal conversion, establishes this line as a valuable resource for investigating the relationship between cellular aging and HD pathogenesis.

It has been previously shown that some short peptides are involved in various cellular processes, such as transcription modulation and regulation of differentiation mechanisms. In particular, the effect of peptides on the neuronal differentiation of human periodontal ligament stem cells has been demonstrated. The goal of this study was to assess the effect of KED, EDR, and AEDG short peptides in stimulating the transdifferentiation of fetal MSCs into induced neuronal cells and prevention of their senescence. We applied a novel in vitro technique for neuronal cell generation, which combines the use of microRNAs, transcription factors, and small molecules to transdifferentiate fetal mesenchymal stem cells into induced cortical neurons. It was shown that the application of AEDG and KED short peptides at the end of the transdifferentiation process decreases the expression of the cell cycle marker p21 by 15% and beta-galactosidase activity by 1.51–2.4 times. However, short peptides did not affect the expression levels of TUj-1 and LaminB1, whose expression also changes during neuronal differentiation. The experiments indicate the potential of AEDG and KED short peptides as modulators of neurogenesis and geroprotectors and suggest that they can be used as stimulators of neuronal differentiation.

Neurons become more vulnerable to stress factors with age, which leads to increased oxidative DNA damage, decreased activity of mitochondria and lysosomes, increased levels of p16, decreased LaminB1 proteins, and the depletion of the dendritic tree. These changes are exacerbated in vulnerable neuronal populations during the development of neurodegenerative diseases. Glu-Asp-Arg (EDR) and Lys-Glu-Asp (KED), and Ala-Glu-Asp-Gly (AEDG) peptides have previously demonstrated neuroprotective effects in various models of Alzheimer’s disease. In this study, we investigated the influence of EDR, KED, and AEDG peptides on the aging of fibroblast-derived induced neurons. We used a new in vitro cellular model of human neuronal aging based on the transdifferentiation of aged dermal fibroblasts from elderly donors into induced cortical neurons. All peptides promote the arborization of the dendritic tree, increasing both the number of primary processes and the total length of dendrites. Tripeptides have no effect on the activity of mitochondria and lysosomes and the level of p16 protein in induced neurons. EDR peptide reduces oxidative DNA damage in induced neurons derived from elderly donor fibroblasts. Short peptides partially protect induced neurons from age-related changes and stimulate dendritogenesis in neurons. They can be recommended for use as neuroprotective agents.

Huntington’s disease (HD) is an inherited, incurable disease caused by a mutation and expansion of CAG repeats in the HTT gene encoding the huntingtin protein (mHTT). Despite numerous studies conducted on various cellular and animal models, specific mechanisms explaining the biological role of mHTT and its toxicity to striatal neurons have not yet been established, and effective therapy for patients has not been developed. We obtained and characterized a new line of dermal fibroblasts HDDF (Huntington Disease Dermal Fibroblasts) from a patient with a confirmed diagnosis of HD. The growth characteristics of the HDDF line, staining for canonical markers, karyotyping and cell phenotyping were performed. Direct differentiation of fibroblasts from the resulting line into induced striatal neurons was carried out. The new fibroblast line can be used as a cell model to study the biological role of mHTT and various manifestations of HD pathogenesis both in fibroblasts themselves and in induced neuronal cells obtained using reprogramming methods.

A new in vitro model of Huntington's disease (HD) has been developed, based on the direct reprogramming of dermal fibroblasts from patients with the disease into striatal neurons. Direct reprogramming makes it possible to obtain neurons, preserving the epigenetic information inherent in the cells of a specific donor, and, consequently, the age-associated phenotype of the disease. The new model resembles the main histopathological feature of Huntington's disease – the accumulation of aggregates of the mutant huntingtin protein in striatal neurons obtained from the patient's fibroblasts. Conducting experiments on induced neurons from patients in culture, obtained using direct reprogramming, allows us to individually assess the degree of progression of neuropathology and implement a personalized approach in choosing a treatment strategy and selecting drugs for therapy. The in vitro model of Huntington's disease can be used for preclinical drug studies, for the treatment of neurodegenerative diseases and for assessing their effectiveness on neurons of specific patients.

Huntington’s disease (HD) is an incurable hereditary disease caused by expansion of the CAG repeats in the HTT gene encoding the mutant huntingtin protein (mHTT). Despite numerous studies in cellular and animal models, the mechanisms underlying the biological role of mHTT and its toxicity to striatal neurons have not yet been established and no effective therapy for HD patients has been developed so far. We produced and characterized a new line of dermal fibroblasts (HDDF, Huntington’s disease dermal fibroblasts) from a patient with a confirmed HD diagnosis. We also studied the growth characteristics of HDDF cells, stained them for canonical markers, karyotyped these cells, and investigated their phenotype. HDDF cells was successfully reprogrammed into induced striatal neurons via transdifferentiation. The new fibroblast line can be used as a cell model to study the biological role of mHTT and manifestations of HD pathogenesis in both fibroblasts and induced neuronal cells obtained from them by reprogramming techniques.

A new in vitro model of Huntington’s disease (HD) was developed via a direct reprogramming of dermal fibroblasts from HD patients into striatal neurons. A reprogramming into induced pluripotent stem (iPS) cells is obviated in the case of direct reprogramming, which thus yields neurons that preserve the epigenetic information inherent in cells of a particular donor and, consequently, the age-associated disease phenotype. A main histopathological feature of HD was reproduced in the new model; i.e., aggregates of mutant huntingtin accumulated in striatal neurons derived from a patient’s fibroblasts. Experiments with cultured neurons obtained via direct reprogramming make it possible to individually assess the progression of neuropathology and to implement a personalized approach to choosing the treatment strategy and drugs for therapy. The in vitro model of HD can be used in preclinical drug studies.

Huntington’s disease is a hereditary incurable neurodegenerative disease caused by expansion of the polyglutamine tract in exon 1 of the huntingtin gene. Huntingtin is a large protein involved in many cellular processes, such as division, transport and secretion. Mutations in the protein lead to disruption of many cellular processes, including secretion, but differences in the composition of the secretome of cells in normal conditions and in Huntington’s disease remain poorly studied. Since huntingtin is expressed at high levels in peripheral tissues and in the skin, we have focused our attention on the study of secretome produced by dermal fibroblasts. In order to identify differences in secreted factors caused by the huntingtin mutation we used tandem time-of-flight mass spectrometry. Forty-two differential proteins were identified in the secretomes of dermal fibroblasts from healthy donor and patient with Huntington’s disease. We examine several proteins of interest including filamin A, periostin, ACTN4, BASP1, adrenomedullin, HSP70 and 14-3-3, whose expression is associated with processes such as cytoskeletal organization, cell adhesion, proliferation, cell migration, protein binding and regulation of cytoskeletal structure. HSP70 and 14-3-3 have neuroprotective properties, and interestingly, their expression was not detected in the secretome of cells with Huntington’s disease. Thus, it was shown that the set of proteins secreted into the extracellular space by dermal fibroblasts with the Huntington’s disease genotype differs from healthy cells, and the differences in cellular processes (proliferation, migration) observed in these cells in vitro are probably due to differences in the composition of the extracellular matrix which they synthesize.

The modeling of neuropathology on induced neurons obtained by cell reprogramming technologies can fill a gap between clinical trials and studies on model organisms for the development of treatment strategies for neurodegenerative diseases. Patient-specific models based on patients’ cells play an important role in such studies. There are two ways to obtain induced neuronal cells. One is based on induced pluripotent stem cells. The other is based on direct reprogramming, which allows us to obtain mature neuronal cells from adult somatic cells, such as dermal fibroblasts. Moreover, the latter method makes it possible to better preserve the age-related aspects of neuropathology, which is valuable for diseases that occur with age. However, direct methods of reprogramming have a significant drawback associated with low cell viability during procedures. Furthermore, the number of reprogrammable neurons available for morphological and functional studies is limited by the initial number of somatic cells. In this article, we propose modifications of a previously developed direct reprogramming method, based on the combination of microRNA and transcription factors, which allowed us to obtain a population of functionally active induced striatal neurons (iSNs) with a high efficiency. We also overcame the problem of the presence of multinucleated neurons associated with the cellular division of starting fibroblasts. Synchronization cells in the G1 phase increased the homogeneity of the fibroblast population, increased the survival rate of induced neurons, and eliminated the presence of multinucleated cells at the end of the reprogramming procedure. We have demonstrated that iSNs are functionally active and able to form synaptic connections in co-cultures with mouse cortical neurons. The proposed modifications can also be used to obtain a population of other induced neuronal types, such as motor and dopaminergic ones, by selecting transcription factors that determine differentiation into a region-specific neuron.