Calcilytic NPS 2143 Reduces Amyloid Secretion and Increases sAβPPα Release from PSEN1 Mutant iPSC-Derived Neurons

Abstract

Despite numerous efforts and studies over the last three decades, Alzheimer's disease (AD) remains a disorder not fully understood and incurable so far. Development of induced pluripotent stem cell (iPSC) technology to obtain terminally differentiated neurons from adult somatic cells revolutionized the study of AD, providing a powerful tool for modelling the disease and for screening candidate drugs. Indeed, iPSC reprogramming allowed generation of neurons from both sporadic and familial AD patients with the promise to recapitulate the early pathological mechanisms in vitro and to identify novel targets. Interestingly, NPS 2143, a negative allosteric modulator of the calcium sensing receptor, has been indicated as a possible therapeutic for AD. In the present study, we assessed the potential of our iPSC-based familial AD cellular model as a platform for drug testing. We found that iPSC-derived neurons respond to treatment with ␥-secretase inhibitor, modifying the physiological amyloid-␤ protein precursor (A␤PP) processing and amyloid-␤ (A␤) secretion. Moreover, we demonstrated the expression of calcium sensing receptor (CaSR) protein in human neurons derived from healthy and familial AD subjects. Finally, we showed that calcilytic NPS 2143 induced a changing of A␤ and sA␤PP␣ secreted into conditioned media and modulation of CaSR and PSEN1 expression at the plasma membrane of AD neurons. Overall, our findings suggest that NPS 2143 affects important AD processes in a relevant in vitro system of familial AD.

Detection and Functional Evaluation of the P2X7 Receptor in hiPSC Derived Neurons and Microglia-Like Cells

“…To examine the presence and the functionality of the P2X7R on human neuronal cells, the NPCs were terminally differentiated ( Figure 1 ). The neuronal differentiation of the used hiPSCs, as well as the AD phenotype of the PSEN1 mutant hiPSC line, was characterized and published previously by our laboratory ( Ochalek et al, 2017 ; Lo Giudice et al, 2019 ). Here, the neuronal cells were kept in culture for 63 days (TD63) to examine the effects of long-term culturing on the AD pathology manifestation and the expression of the P2X7R.…”

“…Here, we showed the basic characteristics of the used neuronal cells, including the main AD-pathology-related trait—APP-CTF expression. The AD-phenotype of the neuronal cells used in this study was extensively studied and assessed in our previous published work ( Chandrasekaran et al, 2017 ; Ochalek et al, 2017 ; Lo Giudice et al, 2019 ). Previously, we reported the establishment of a hiPSC-based cellular platform able to model in vitro the major pathological events of both the familial and the sporadic form of Alzheimer’s disease in neuronal cell cultures.…”

“…In the present study, hiPSCs, derived from a healthy Caucasian female (35 years old) (here named as Ctrl) ( Ochalek et al, 2017 ), and the PSEN1 (p.Val89Leu) mutant familial Alzheimer’s disease female patient (55 years old) (here named as fAD) ( Nemes et al, 2016 ), which we characterized in our previous studies ( Ochalek et al, 2017 ; Lo Giudice et al, 2019 ) were used for neuronal and microglia (control cell line) differentiation. Human hiPSCs were cultured on BD Matrigel™ matrix (BD Biosciences, Franklin Lakes, NJ, United States) with mTeSR™1 medium (Stem Cell Technologies, Vancouver, Canada), using Gentle Cell Dissociation Reagent for passages, according to the manufacturer’s instruction.…”

“…hiPSCs are particularly useful in modeling otherwise hardly accessible tissues, like the human brain ( Chambers et al, 2009 ; Nicholas et al, 2013 ). Protocols for the generation of neurons and astroglia from hiPSC are readily available and utilized routinely in several laboratories worldwide, including ours ( Zhou et al, 2016 ; Chandrasekaran et al, 2017 ; Ochalek et al, 2017 ; Lo Giudice et al, 2019 ). However, microglia generation from hiPSCs has been problematic for a long time due to the microglia’s unique yolk sac origin.…”

Contribution of Human Pluripotent Stem Cell-Based Models to Drug Discovery for Neurological Disorders

“…Both have received a great deal of attention in an effort to develop new pharmacological strategies [ 138 ]. Modelling of familial early-onset AD demonstrated that neurons differentiated from patient-derived iPSCs successfully recapitulated characteristic AD phenotypes, including the formation of Aβ aggregates and neurofibrillary tangles, which can be reversed by candidate pharmacological treatments, confirming the value of the model in the search for new pharmacological approaches [ 139 , 140 , 141 , 142 , 143 ]. Aβ accumulation and tau hyperphosphorylation were also reported using iPSCs derived from sporadic cases [ 144 ].…”