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Review
. 2021 Apr 27;22(9):4596.
doi: 10.3390/ijms22094596.

iPSCs: A Preclinical Drug Research Tool for Neurological Disorders

Gabriele Bonaventura  1 , Rosario Iemmolo  1 , Giuseppe Antonino Attaguile  2 , Valentina La Cognata  1 , Brigida Sabrina Pistone  1 , Giuseppe Raudino  3 , Velia D'Agata  4 , Giuseppina Cantarella  2 , Maria Luisa Barcellona  5 , Sebastiano Cavallaro  1
Affiliations
Review

iPSCs: A Preclinical Drug Research Tool for Neurological Disorders

Gabriele Bonaventura et al. Int J Mol Sci. .

Abstract

The development and commercialization of new drugs is an articulated, lengthy, and very expensive process that proceeds through several steps, starting from target identification, screening new leading compounds for testing in preclinical studies, and subsequently in clinical trials to reach the final approval for therapeutic use. Preclinical studies are usually performed using both cell cultures and animal models, although they do not completely resume the complexity of human diseases, in particular neurodegenerative conditions. To this regard, stem cells represent a powerful tool in all steps of drug discovery. The recent advancement in induced Pluripotent Stem Cells (iPSCs) technology has opened the possibility to obtain patient-specific disease models for drug screening and development. Here, we report the use of iPSCs as a disease model for drug development in the contest of neurological disorders, including Alzheimer's (AD) and Parkinson's disease (PD), Amyotrophic lateral Sclerosis (ALS), and Fragile X syndrome (FRAX).

Keywords: AD; ALS; FRAX; PD; drug development; iPSCs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of cells reprogramming and neuronal differentiation in vitro. (a) A schematic overview of human iPSC line generation from fibroblasts. Fibroblasts are transduced with Sendai Vector (one of the non-integrating method) carrying OCT4, KLF4, SOX2, and MYC. On day 12, conditionally reprogrammed cells start to emerge in colonies expressing high levels of KOS, KLF4, and c-MYC mRNAs. On day 30, reprogrammed embryo bodies express the pluripotent stem cell markers TRA1-60 and TRA1-81, as revealed by immunofluorescence assays, and are ready for picking-up and expansion. (b) Representative scheme highlighting the timing of human iPSCs differentiation towards motor neuronal lineage. During neuralisation, cell morphology changes from expanding colonies to neural rosettes (NE). Cells acquire reduced cell soma area and thin extended projections connecting adjacent cells, finally differentiating into motor neurons-like cells. At the end of differentiation protocol, iPSC-derived neurons express specific neuronal markers (TUJ-1, SMI32, MAP2, ChAT).
Figure 2
Figure 2
iPSC-based drug testing to treat Alzheimer’s Disease. Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, characterized by an abnormal deposition of extracellular amyloid plaques. Amyloid plaques are made of insoluble β-Amyloid (Aβ) peptide deposition produced by sequential cleavages of amyloid precursor protein (APP) by b-site APP cleaving enzyme 1 (BACE1), β-and γ-secretase. Drug testing efforts conducted on AD-derived iPSC allowed to identify an anti-Aβ cocktail, composed by a mix of topiramate, cromolyn and bromocriptine, able to reduce Aβ deposition and plaques formation. Other studies have demonstrated the β-secretase inhibitor IV (BSI), γ-secretase inhibitor XXI/Compound E and sulindac sulfide efficacy in partially or fully blocked Aβ production. Illustrations used elements from Servier Medical Art (www.servier.fr/servier-medical-art).
Figure 3
Figure 3
iPSC-based drug testing to treat Amyotrophic Lateral Sclerosis. ALS is considered a complex multifactorial and genetic disorder characterized by protein aggregation, mitochondrial dysfunction, oxidative stress and excitotoxicity, leading to motor neuron apoptosis. Drug testing has been conducted in order to rescue iPSC-derived affected motor neurons. The potassium channel blocker 4-Aminopyridine (4AP) or the antiepileptic ezogabine demonstrated their ability to reduce neuronal damage by restoring hypoexcitable phenotypes in FUS, SOD1 or C9ORF72 mutant iPSC-derived motor neurons. Tubastatin A and ACY-738, two specific histone deacetylase 6 (HDAC6) inhibitors, restore mitochondrial transport defects along FUS mutated neuronal axons. Natural peptides, such as PACAP or VIP, were shown to rescue iPSC-derived motor neurons from apoptosis, restoring mitochondrial activity and neurite outgrowth. Illustrations used elements from Servier Medical Art (www.servier.fr/servier-medical-art).
Figure 4
Figure 4
iPSC-based drug testing to treat Parkinson’s Disease. The main pathological characteristics of PD are the presence of Lewy bodies (aggregation and accumulation of the protein α-synuclein), which cause mitochondrial disfunctions, endoplasmatic reticulum (ER) stress and Golgi degradation, leading to cell death in the brain’s basal ganglia. Human iPSC-derived dopaminergic models have facilitated the study of PD, providing amenable systems for drug discovery as well. Promising results derive from drugs targeting the glucocerebrosidase (GCase) pathway. Inactive GCase (in red) can be restored in active GCase (in green) directly using GCase chaperones NCGC758, NCGC607 and novel small molecules, such as S-181, or indirectly using Quinazoline modulators able to trig GCase activator. These compounds reduce lysosome accumulation of α-synuclein in both wildtype and mutant GCase patient-specific iPSC-derived dopaminergic neurons. SNCA aggregates can also be directly targeted by small molecules NPT100-18A, NPT100-14A and their derivatives, which are able to revert the degenerative phenotype. Different drugs, such as isoxazole, coenzyme Q10, rapamycin and GW5074 (a LRRK2 kinase inhibitor) have been tested in iPSC-DA models showing effects in increasing mitochondria respiration and biogenesis. The stress of ER is another drug target to treat PD. A novel unfolded protein response (UPR) modulator, azoramide restore ER function in PLA2G6 D331Y mutant DA neurons. Illustrations used elements from Servier Medical Art (www.servier.fr/servier-medical-art).
Figure 5
Figure 5
iPSC-based drug testing to treat Fragile X Syndrome. FRAX is a monogenic disorder related to a loss-of-function mutation type of FMR1 gene containing an expansion of the cytosine-guanine-guanine (CGG) triplets repeat located within the 5′ untranslated region (UTR) of the gene. Alleles with more than 200 repeats, considered full mutated alleles (FM), present the FMR1 promoter hypermethylated, which prevents the expression of FMR1 due to epigenetic gene silencing. iPSCs provide a valuable instrument for developing gene therapy-based approaches to FRAX. Different chemical compounds have been studied to remove epigenetic marks. To date, two approaches have been tested. The first one investigated the effect of chromatin remodeling compounds, such as trichostatin-A (TSA), 5-azacytidine (5-azaC), and 5-aza-2′-deoxycytidine (5-aza-dC). These compounds lead to the loss of DNA methylation by the inhibition of DNA methyltransferases (DNMT), restoring FMR1 expression in FRAXA iPSCs and their neural derivatives. The second approach was based on dCas9-Tet1/single guide RNA tool, a new developed DNA methylation editing technology that induces demethylation of the CGG expansion, switches the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, and restores a persistent expression of FMR1 in FRAXA iPSCs. Illustrations used elements from Servier Medical Art (www.servier.fr/servier-medical-art).
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