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Learn more: PMC Disclaimer | PMC Copyright Notice Alzheimers Dement . 2026 Apr 16;22(4):e71281. doi: 10.1002/alz.71281 Search in PMC Search in PubMed View in NLM Catalog Add to search Evaluating the peripheral nervous system pathology of Alzheimer's disease utilizing a functional human NMJ microphysiological system Akhmetzada Kargazhanov Akhmetzada Kargazhanov 1 Hybrid Systems Lab, NanoScience Technology Center, University of Central Florida, Orlando, Florida, USA Find articles by Akhmetzada Kargazhanov 1 , Romy Aiken Romy Aiken 1 Hybrid Systems Lab, NanoScience Technology Center, University of Central Florida, Orlando, Florida, USA Find articles by Romy Aiken 1 , Kenneth Hawkins Kenneth Hawkins 1 Hybrid Systems Lab, NanoScience Technology Center, University of Central Florida, Orlando, Florida, USA Find articles by Kenneth Hawkins 1 , Rafael Lopez Rafael Lopez 1 Hybrid Systems Lab, NanoScience Technology Center, University of Central Florida, Orlando, Florida, USA Find articles by Rafael Lopez 1 , Ahmad Nawaz Ahmad Nawaz 1 Hybrid Systems Lab, NanoScience Technology Center, University of Central Florida, Orlando, Florida, USA Find articles by Ahmad Nawaz 1 , Gaurav Srivastava Gaurav Srivastava 1 Hybrid Systems Lab, NanoScience Technology Center, University of Central Florida, Orlando, Florida, USA Find articles by Gaurav Srivastava 1 , Chase Miller Chase Miller 2 Hesperos Inc. Orlando, Florida, USA Find articles by Chase Miller 2 , Will Bogen Will Bogen 2 Hesperos Inc. Orlando, Florida, USA Find articles by Will Bogen 2 , Christopher Long Christopher Long 2 Hesperos Inc. Orlando, Florida, USA Find articles by Christopher Long 2 , David Morgan David Morgan 3 Department of Translational Neuroscience, Michigan State University College of Human Medicine, Grand Rapids, Michigan, USA Find articles by David Morgan 3 , Xiufang Guo Xiufang Guo 1 Hybrid Systems Lab, NanoScience Technology Center, University of Central Florida, Orlando, Florida, USA Find articles by Xiufang Guo 1 , James Hickman James Hickman 1 Hybrid Systems Lab, NanoScience Technology Center, University of Central Florida, Orlando, Florida, USA 2 Hesperos Inc. Orlando, Florida, USA Find articles by James Hickman 1, 2, ✉ Author information Article notes Copyright and License information 1 Hybrid Systems Lab, NanoScience Technology Center, University of Central Florida, Orlando, Florida, USA 2 Hesperos Inc. Orlando, Florida, USA 3 Department of Translational Neuroscience, Michigan State University College of Human Medicine, Grand Rapids, Michigan, USA * Correspondence , James Hickman, Hybrid Systems Lab, NanoScience Technology Center, University of Central Florida, 12424 Research Parkway, Suite 400, Orlando, FL 32826, USA. Email: [email protected] ✉ Corresponding author. Received 2025 Oct 20; Accepted 2025 Dec 22; Collection date 2026 Apr. © 2026 The Author(s). Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association. This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. PMC Copyright notice PMCID: PMC13084706 PMID: 41988945 Abstract INTRODUCTION Alzheimer's Disease (AD) is a central nervous system (CNS) neurodegenerative disease leading to dementia, but can also show symptoms of motor deficits. It is not clear whether the peripheral motor deficits in AD are derived from upstream centers or intrinsic to the neuromuscular circuit. This study developed a model to evaluate the neuromuscular pathology of familial AD (fAD) in a functional neuromuscular junction (NMJ) system. METHODS The fAD iPSC motoneurons (MNs), together with healthy iPSC skeletal myoblasts (SKM), were adapted into a dual chamber NMJ system. The formation and function of the NMJs formed were evaluated utilizing clinically translatable readouts. RESULTS Functional analysis indicated that NMJs formed with fAD MNs showed severe (PSEN1 A246E) to moderate (APP K595N/M596L) deficiencies in NMJ function. DISCUSSION These findings confirmed that fAD mutations lead to NMJ deficiencies, supporting that motor deficits can be induced independently from cognitive deficits. Keywords: fAD NMJ pathology, familial Alzheimer's disease, microphysilogical system, motoneurons, neuromuscular junction, peripheral nervous system Highlights This study demonstrated development of NMJ pathology in familial AD (fAD) in an isolated human NMJ model. First human‐based MPS model for simulating and investigating NMJ pathology in AD. Existing AD therapeutics induced no significant functional improvement in fAD NMJ. Potentially other therapeutics are needed to correct the fAD NMJ deficits. 1. BACKGROUND Alzheimer's Disease (AD) is a major cause of dementia and is ranked as the 7 th leading cause of mortality in the US and 1/3 rd of those over 65 have dementia when they die. 1 Although AD is classified as a disorder of cognitive function, clinical data suggests that patients who are diagnosed with AD also show symptoms of motor deficits prior to manifestation of cognitive impairment. 2 From a clinical study conducted at the Tokyo Medical University, it was found that patients diagnosed with early AD showed muscle strength loss, lower gait speed, and reduction in gait speed was directly correlated with the degree of cognitive impairment. 3 A longitudinal study of 12 years conducted by the UK Biobank, found that participants who developed dementia had significantly lower gait speed and significantly weaker grip strength compared to the rest of the cohort at the baseline of the study. 4 In a separate clinical study conducted at the Shanghai Mental Health Center, it was determined that median and common peroneal motor nerve conduction velocity was reduced in patients with mild cognitive impairment and reduced significantly in AD patients compared to the control group. 5 Impairments in the neuromuscular system, that precede cognitive impairment, have been observed in mouse AD models, such as deficiencies in NMJ function, muscle strength loss, peripheral nerve dysfunction, and altered conduction velocity/efficiency for skeletal muscle. 6 , 7 , 8 It can therefore be hypothesized that AD is a systemic disorder that not only affects brain cognition but also the neuromuscular system. Therefore, a parallel strategy of investigating the mechanisms of neuromuscular dysfunction in AD is needed for understanding the entirety of AD pathology. Currently, all AD drugs are designated to alleviate cognitive or psychiatric impairment, but no drugs are designed to improve neuromuscular parameters during AD. 9 An additional constraint on the development of AD therapeutics is the heavy reliance on animal models which may not exactly replicate the pathogenesis of a human disease since there is a significant difference between humans and animals in physiology and development. 10 , 11 , 12 New technologies such as human induced pluripotent stem cells (iPSCs) bypass such interspecies gaps so that patient‐specific treatments can be developed, especially for rare genetic diseases. 13 The iPSC‐derived cells can be integrated into functional in vitro assays such as Bio‐MEMS and Human‐on‐a‐Chip systems. A functional human cantilever assay has been developed that investigated the performance of iPSC‐derived skeletal muscle. 14 A functional in vitro assay has also been developed that is being used to investigate the performance and integrity of NMJs formed between iPSC‐derived motoneurons and skeletal muscle to study neuromuscular system pathologies such as amyotrophic lateral sclerosis (ALS), myasthenia gravis (MG), and Charcot Marie Tooth (CMT) Disease. 15 , 16 , 17 , 18 Its extension to AD in this report opens a new paradigm in Alzheimer's investigations and would parallel the in vitro AD long term potentiation (LTP) microphysiological system (MPS) that has been used for evaluation of CNS therapeutics targeting cognitive symptoms. 19 , 20 Using efficacy data from one of our MPS systems, Sanofi filed the first IND that led to the FDA authorizing a Phase II clinical trial ( NCT04658472 ) and this has led to a two‐armed Phase III clinical trial ( NCT06290141 and NCT06290128 ). Availability of the Bio‐MEMS NMJ functional assay and iPSCs from familial AD (fAD) patients enables investigation of the hypothesis of whether there is neuromuscular pathology in fAD. To develop a functional NMJ model for fAD, two iPSC lines carrying different heterozygous fAD mutations: PSEN1 A246E derived from a fAD patient, and iso‐APP (K595N/M596L), the “Swedish” mutation generated through isogenic editing, were utilized. The formation and function of NMJs between MNs harboring these fAD mutations and skeletal muscle derived from iPSCs of healthy donors (wild‐type, WT) were investigated in a two chamber MPS. In addition, the efficacy of two therapeutics commonly prescribed during the early stages of AD, memantine and galantamine, were investigated in this fAD NMJ platform. 2. METHODS 2.1. Motoneuron differentiation from iPSCs Human iPSCs from a healthy donor (ND41865 WTA) were obtained from Coriell Institute, Camden, NJ. Human iPSCs with a knock‐in“Swedish” APP mutation (CO0002‐01‐CS‐003) and the parent isogenic control iPSCs (CO0002‐01‐SV‐003) were obtained from the New York Stem Cell Foundation. Human iPSCs from a fAD donor with the PSEN1 A246E (CS40iFAD‐nxx PSEN1) mutation was obtained from Cedars‐Sinai. Human iPSC to MN differentiation was completed by following an established protocol. 16 , 21 RESEARCH IN CONTEXT Systematic review : Literature of human clinical studies demonstrate that patients with AD suffer from loss of muscle mass, leg and arm strength and gait speed even before the onset of CNS symptoms, suggesting presence of AD neuromuscular pathology. Literature from animal studies indicates that existence of neuromuscular AD pathology, but it is not clear whether the motor deficit is developed through its association with the cognitive pathology or an independent phenotype. The investigation of human AD neuromuscular pathology requires development of a human centric neuromuscular model. Interpretation : This study aimed to develop an in vitro model to evaluate the neuromuscular pathology of AD by integrating motoneurons (MNs) differentiated from patient induced pluripotent stem cells (iPSCs) harboring fAD mutations into a functional neuromuscular junction (NMJ) system. Systematic phenotypic analysis of these human fAD MN NMJ models has uncovered important mechanistic insights as the motor deficits correlate with those observed in AD patients. The evaluation of existing AD therapeutics in this model indicated no significant improvement, suggesting other therapeutics are needed to correct the fAD MN NMJ deficits and peripheral deficits in general. Future directions : fAD skeletal muscle (SKM) can be integrated into the existing functional NMJ and SKM models, which can provide further mechanistic insight into human neuromuscular fAD pathology. Novel therapeutics of repurposed drugs can be screened in this model system. 2.2. Fabrication, assembly, and coating of Bio‐MEMs NMJ assay Polydimethylsiloxane (PDMS) chambers were designed according to the methods and dimensions described in previous publications. 21 Chambers were fabricated by curing PDMS (Dow SYLGARD™ 184 Silicone Encapsulant Clear 3.9 kg Kit) over SU‐8 molds at 65°C for 24 h. After cutting, chambers were soaked in 70% IPA overnight, followed by rinsing with 3 volumes of 70% IPA. Later, chambers were sterilized in 70% IPA and air‐dried overnight in a biosafety cabinet. Chambers were activated by plasma treatment for 45 s with oxygen pressure at 750 mTorr. After plasma treatment, 22 × 22 mm 2 glass coverslips coated with N‐1 [3‐(trimethoxysilyl) propyl] diethylenetriamine (DETA), that was heated at 80°C, were assembled onto the plasma‐activated PDMS chambers by applying pressure with plastic tweezers across the perimeter of the DETA coverslip‐PDMS chamber contact including the tunnel portion. The assembled chambers were incubated at 80°C for 5 min and later pressure was applied with plastic tweezers around the perimeter of the DETA coverslip‐PDMS chamber contact including the tunnel portion. The assembly was later UV sterilized for 10 min in a biosafety cabinet. The next day the MN side was coated with 10 µg/mL of Laminin (Fisher Scientific) and 10 µg/mL of Fibronectin (Sigma Aldrich) resuspended in phosphate buffer saline (PBS). Also, the SKM side was coated with 1 µg/mL of rat tail collagen 1 (Fisher Scientific) and 133 µg/mL of human skin elastin (Elastin Products Company, Inc). The ECM in the NMJ chambers was incubated at 37°C for 1 h. Later, the ECM solution was removed and replaced with PBS. The insulation between chambers was measured by a voltohmmeter (World Precision Instrument) prior to myoblast plating. Systems with TEER measurements below 7000 Ω were eliminated. 2.3. Plating myoblasts and MNs in the Bio‐MEMs NMJ chamber The myoblasts were differentiated from iPSCs of a healthy donor according to the protocol described in a previous publication. 22 The myoblasts were plated in DMEM with 1000 mg/L D‐Glucose (Stemcell Technologies) supplemented with Myocult (Stemcell Technologies) at 200 cells/mm 2 from passage 6. Myoblasts were allowed to grow until 70%–100% confluency in the same medium, which was followed by a half‐medium change to DKHI (DMEM/F12 (Thermo Fischer Scientific), 15% Knockout Serum (Fischer Scientific), 1% non‐essential amino acids (Thermofisher), 1% Antibiotic/Antimycotic (Thermofisher), 0.1 mM Mercaptoethanol (Thermofisher), 1 ng/mL HGF (Perpotech), 2 ng/mL IGF (Millipore Sigma) and later supplemented with 100 nM dexamethasone (Millipore Sigma). Two days later, the SKM chamber was fed by a half medium change with NBactive4 (Transnetyx) prior to HMN plating. MNs were plated at 1500 cells/mm 2 in a motoneuron medium. 2.4. Functional NMJ testing On day 10 after MN plating, NMJ systems were assembled into the acrylic housing required for functional testing (Hesperos). Silver wire that was 0.025 inches in diameter (AM Systems) was chlorinated in 100% bleach (Clorox) for 1 h and later cut and coiled into electrodes. On day 11 from MN plating, 4 chlorinated electrodes were assembled onto the PCB board adapted to the NMJ housings which could be connected to the current stimulator PCB board. Later the PCB board with chlorinated electrodes was assembled on the housing with the NMJ system, with 2 electrodes inserted into each MN and SKM chamber. NMJ systems with attached electrode‐PCB assembly were connected to the stimulator PCB and placed under the microscope (Zeiss Hal 100). Functional NMJ testing videos were recorded with a Hamamatsu digital camera (Model C8484‐05G) with high‐speed acquisition of 50 frames per second. A LabVIEW program was used for the functional NMJ testing recording and electrical pulse stimulator PCB control. The contracting myotubes were identified based on the phase contrast differentials. During NMJ testing electrical stimulations of 1 V were applied either to the MN chamber or SKM chamber at the frequencies 0.33 Hz, 0.5 Hz, 1 Hz, 2 Hz, and 4 Hz for 15 s per frequency. A Python‐based graphical interface was used to analyze recordings generated during the NMJ testing allowing selection of Regions Of Interest, analysis of pixel subtraction magnitudes, fidelity and fatigue index. During the functional testing the PCB heater control was set to the temperature of 20°C. For the drug treatment experiment, memantine (Cayman Chemical) were prepared in MN medium and administered to the MN chambers starting from day 13 from MN plating. Galantamine (Cayman Chemical) was resuspended in DMSO and then prepared in MN medium when administered to MN chamber and SKM medium when administered to SKM chamber starting from day 13 from MN plating. 2.5. Electrophysiological analysis of fAD MNs The electrophysiological properties of iPSC‐derived MNs were analyzed by whole‐cell patch clamp analysis as in the previous publications. 21 , 23 Borosilicate glass pipettes (BF 150‐86‐10; Shutter Instrument Company) with a resistance of 6‐10 MΩ were made using a Shutter P97 pipette puller (Shutter Instrument Company). The intracellular solution used in the patch clamp pipette contained 1 mM EGTA, 140 mM K‐gluconate, 2 mM MgCl 2 , 2 mM Na 2 ATP and 10 mM HEPES (pH 7.2). The MN medium was supplemented with 10 mM HEPES (pH 7.2) and was used as the extracellular solution for all patch experiments. Cultured MNs maintained on round glass coverslips with a diameter of 18 millimeter and coated with DETA were placed on the stage of the upright microscope (Zeiss Axioscope 2FS Plus). After the formation of a giga‐Ω seal and membrane puncture of the MNs the capacitance was compensated. All current‐clamp and voltage‐clamp recordings were taken using a Multiclamp 700A amplifier (Axon Instruments). Signals during the patch clamp recordings were filtered at 3 kHz and sampled at 20 kHz using a Digidata 1322A interface (Axon Instruments). The recording of the data and analysis was performed using pClamp8 software (Axon Instruments). The membrane potentials were adjusted by subtracting 15 mV using pClamp8 software. Sodium and potassium currents were recorded in voltage‐clamp mode. Induced repetitive firing was measured during 1 s of depolarizing current injections from a −70 mV holding potential in current clamp mode. Spontaneous MN activity was recorded in GAP free mode. 2.6. Immunocytochemistry MNs were fixed at RT with 4% paraformaldehyde in a 7.2 pH PBS for 15 min. Mummified MNs were incubated 2 times at RT for 5 min with PBS followed by permeabilization with 0.1% Triton X‐100 in PBS for 10 min. The MNs were blocked for 1 h at RT in a blocking buffer consisting of 3% bovine serum albumin and 0.05% Tween‐20 in PBS. MNs were incubated at 4°C overnight with primary antibodies diluted in the blocking buffer. Next day, MNs were incubated in PBS for 3 × 10 min at RT, followed by incubation with secondary antibodies diluted in the blocking buffer for 2 h at RT in a dark‐room. Later, MNs were incubated in PBS for 3 × 10 min at RT in a dark room. Coverslips with MNs were mounted onto the microscope slides using a ProLong™ Diamond Antifade Mountant with DAPI (Thermofisher) and stored in a dark box at 4°C. The imaging was done using a Zeiss Axioscope confocal microscope coupled with Velocity Software (Quorum Technologies). Prior to MN imaging the exposure times were adjusted by imaging MNs stained with secondary antibody only controls. Primary antibodies used in this study: MAP2 polyclonal antibody (1:1000 dilution, PA1‐10005, Thermofisher), Recombinant Anti‐Choline Acetyltransferase antibody (1:300 dilution, Ab181023 , Abcam), Purified anti‐Neurofilament Marker SMI312 (pan axonal, cocktail) Antibody (1:300 dilution, 837904, Biolegend), RAB5 Polyclonal Antibody (1:300 dilution, PA3‐915, Thermofisher), Myosin heavy chain 1 Polyclonal Antibody (1:300 dilution, PA5‐117077, Thermofisher) and Alpha‐Bungarotoxin Conjugate (1:100 dilution, B35450 , Thermofisher). Secondary antibodies used in this study: donkey anti‐mouse 567 (1:250 dilution, Thermofisher), donkey anti‐chicken 647 (1:250 dilution, Thermofisher), donkey anti‐rabbit 488 (1:250 dilution, Thermofisher) and donkey anti‐chicken 488 (1:250 dilution, Thermofisher). 2.7. Statistical analysis The error bars represent standard error mean (SEM). One‐way ANOVA was performed with the appropriate multiple comparisons post‐test, either Dunnett's test or Sidak's test. For the drug treatment experiment, two‐way ANOVA was performed with Tukey multiple comparisons post‐test. Each NMJ chamber was a biological replicate (r) for functional analysis. Each individual MN was a biological replicate (r) for electrophysiology and immunocytochemistry. The N represents the number of times (batches) the experiment was repeated. Statistical analysis was performed during the initial stages in MS Excel and later in GraphPAD Prism 10. 3. RESULTS 3.1. Characterization of AD‐MNs from patient‐derived iPSCs Two iPSC lines carrying different heterozygous fAD mutations, PSEN1 A246E and APP (K595N/M596L), were selected and utilized. The fAD mutations were compared to two human control lines derived from two healthy subjects, one of which is the parental line for generating the iso‐APP. To develop a functional NMJ model for fAD, MNs were differentiated from iPSCs harboring these fAD mutations, and the two control iPSC lines, by utilizing an established MN protocol (Figure 1 ). 21 , 23 The identity of the differentiated MNs were confirmed by immunocytochemistry demonstrating the expression of the neuronal marker microtubule‐associated protein (MAP2) and MN‐specific marker choline acetyl transferase (CHAT) (Figure 2 ). FIGURE 1. Open in a new tab Plating timeline of the NMJ co‐culture with the schematic representing the NMJ Bio‐MEMs system. (A) Timeline of the NMJ experiment; (B) Example images from the experimental process. FIGURE 2. Open in a new tab Characterization of iPSC‐AD‐MNs differentiated from IPSCs derived from fAD patients. CHAT and MAP2 immunocytochemistry images of healthy control MNs, PSEN1 MNs, Isogenic Control MNs, and APP MNs at DIV 11. scale bar 50 µm. The electrophysiological properties of the iPSC‐MNs were investigated by whole‐cell patch clamp electrophysiology (Figure 3 ). MNs differentiated from the fAD iPSC lines (fAD MNs) produced action potentials (APs) of amplitude similar to those of the WT MNs, indicating their electrophysiological competency (Figure 3Ei ). Neuronal hyperexcitability has been observed clinically for certain mutations in the early stages of AD, where the degree of hyperexcitability positively correlated with the severity of AD in later stages. 24 , 25 Based on the patch clamp analysis, hyperexcitability was only suggested in PSEN1 MNs at DIV 12 by its significantly more positive resting membrane potential compared to the WT control (Figure 3Eii ). Interestingly, both PSEN1 and APP MNs showed hypoexcitability at DIV 19 compared to the WT control, based on a significantly lower sodium to potassium current ratio for PSEN1 MNs (Figure 3Eiii ) and the substantially lower rate of firing with current clamp for the APP MNs (Figure 3Eiv ). Based on findings from this study the pattern of MN excitability for the PSEN1 mutation was hypoexcitable in week 1, hyperexcitable in week 2 and hypoexcitable during the later analysis in week 3; while MNs with APP mutation were only hypoexcitable during the later stages of the analysis and generally showed patterns of excitability similar to WT control in all parameters (Figure 3E ). Somewhat similar to the PSEN1 mutation, the pattern of MN hyperexcitability followed by a hypo‐excitability is observed in another neurodegenerative disease investigated with this model, amyotrophic lateral sclerosis (ALS). 26 It is worth noting that in ALS, MNs that show hyperexcitability early on contribute to the pathology later after disease onset. 27 Gap‐free recordings of fAD MNs indicated no significant difference in spontaneous activity compared to WT MNs (Figure S1A,Bi ). Additionally, APP MNs showed significantly higher membrane capacitance compared to WT MNs at DIV 12 and 19 (Figure S1Bii ). Also, PSEN1 MNs showed significantly higher membrane resistance compared to WT MNs at DIV 19 (Figure S1Biii ). FIGURE 3. Open in a new tab Electrophysiological properties of iPSC‐AD‐MNs analyzed by patch clamp on days 6, 12 and 19. (A) Representative phase images of iPSC‐MNs (DIV 12) during the patch clamp analysis; (B) Representative traces of iPSC‐MNs (DIV 12) during the patch clamp analysis, under current clamp conditions (Induced repetitive firing); (C) Representative traces of iPSC‐MNs (DIV 12) during the patch clamp analysis, under voltage clamp conditions (Na, K currents); (D) Representative traces of Ipsc‐MNs (DIV 12) during the patch clamp analysis, under current clamp conditions (Induced AP); (E) Quantification of the patch clamp analysis of iPSC‐MNs (DIV 6, 12 and 19): (i) Induced action potential; (ii) Resting membrane potential; (iii) Ratio of Na+/ K+ currents; (iv) Induced repetitive firing; N = 3 (Number of times experiment was repeated); Data collected from more than 3 independent biological replicates per condition; One‐way ANOVA, Dunnett Multiple comparison. ( p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***)). Recently there have been hypotheses proposed regarding the mechanism of AD that reconciles all classical AD hypotheses under one umbrella. 28 , 29 Based on genetic studies, clinical information, and various studies on fAD and sAD, it has been proposed that the dysregulated endo‐lysosomal (ELN) network functions as a potential active player in AD pathogenesis. 28 , 29 It has been previously shown that enlargement of early endosomes has been observed in the brains of patients diagnosed with AD. 30 , 31 , 32 Studies have shown that APP processing occurs in various endo‐lysosomal compartments linking ELN functions to the amyloid cascade hypothesis. 33 Several studies have shown that impaired endosomal recycling contributes to tauopathy, excitotoxicity, and microglial AD pathology. 34 , 35 , 36 , 37 From in vitro studies, it has been found that neurons with either the PSEN1 A246E or Swedish APP mutations experience enlargement of early endosomes (Rab5 positive), which occurs prior to the accumulation of amyloid plagues. 38 Rab5 immunocytochemistry analysis was conducted on the fAD MNs where Rab5‐positive granules were observed, indicative of the enlarged early endosomes (Figure 4A ). A higher number of Rab5 positive granules in PSEN1 and APP MNs occurred compared to the WT control on days 11 and 17 (Figure 4B ). FIGURE 4. Open in a new tab Rab5 Immunocytochemistry of AD IPSC‐HMNs. (A) Representative images of Rab5, SMI312, and MAP2 stains of AD IPSC‐HMNs at DIV 11, scale bar 50 µm; (B) Number of Rab5 granules per neuron (white arrow used to show example); N = 2 (Number of times experiment was repeated), Data collected from more than 3 independent replicates per condition. One‐way ANOVA, Dunnett Multiple comparisons. ( p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***)). 3.2. Functional characterization of NMJs containing fAD‐MNs Differentiated fAD MNs were integrated into the established functional NMJ model, where co‐culture was adapted into the dual chamber system with microtunnels connecting the skeletal muscle and MN chambers (Figure 1B ). 21 , 39 These microtunnels enable passage of axons from the MN chamber into the skeletal muscle chamber allowing NMJ formation. Since the MN and SKM chambers are chemically and electrically isolated, establishment of dose‐response curves to drugs can be determined independently in each chamber (Figure 1B ). 21 , 39 The NMJ chambers were placed under a microscope connected to a motion capture camera for the skeletal muscle chamber. 21 , 39 Chlorinated silver electrodes connected to a stimulator were placed into the MN chamber and electrical pulses of 1 V were delivered at frequencies of 0.33, 0.5, 1, and 2 Hz. 21 The 2 chambers were electrically isolated with an estimate of less than 1% electrical leakage between chambers, far below the intensity required to directly stimulate muscle contraction (approximately 1–2 V), therefore ensuring the muscle contractions upon MN stimulation were all induced through the NMJ synapse. 21 , 39 Electrical stimulations were applied to the MN chamber, and myotube contractions were detected and monitored by video recordings of pixel differentials (Figure 1B ). 21 , 39 Four parameters were analyzed to investigate the functional pathology of the fAD NMJ systems by stimulating the MN chamber (indirect stimulation). The first parameter was the number of NMJs observed prior to a series of pulse stimulations, which indicates the number of NMJs formed between MNs and skeletal muscle. 21 , 39 The average number of NMJs was estimated by applying single electrical stimulations to the MN chamber and quantifying the number of myotubes that contracted in correspondence to the stimulations. 21 The second parameter was fidelity, reflecting a percentage of the MN stimulations that successfully induced muscle contractions at increasing stimulation frequencies. 21 , 39 The missing muscle contractions upon MN stimulation indicate NMJ deficiencies, such as axonal transport impairment or defects at the synapse. 21 , 39 The third parameter, fatigue index, reflects the decline of myotube tension over time under tetanic conditions at high electrical stimulation frequency (2 Hz). 21 , 39 Normally, myotube tension under tetanus contraction is elevated due to the summation effect, caused by incremental Ca 2+ accumulation inside the sarcolemma under high‐frequency stimulation. 21 , 39 However, due to fatigue of NMJ function caused by the decline of MN stimuli, failure of the MN‐muscle synaptic apparatus, or muscle exhaustion, the tetanus will decline over time, which is quantified as the fatigue index. 21 , 39 To measure NMJ fatigue index, muscle contractions from the indirect traces at 2 Hz MN stimulation were analyzed. 21 , 39 For muscle contraction traces demonstrating tetanus, the peak amplitude of muscle contraction and area under the tetanus curve were analyzed using a Python script, summarized as: Fatigue index = 1 − [Area Under Curve/ (Peak contraction amplitude * Time)]. 21 , 39 The last parameter monitored was NMJ stability, the percentage of NMJ numbers detected following all stimulation protocols after testing divided by the NMJ numbers detected before the initial stimulation protocol. 21 , 39 The NMJ stability is the metric that measures the percentage of NMJs that remained after electrical stimulation protocols, which represents the measure of how well NMJ function was sustained. 21 In the NMJ experiments for this study, myoblasts differentiated from WT iPSCs were plated into the skeletal muscle chamber and expanded to 70%–80% confluency (Figure 1A ). 21 Then, the medium in the skeletal muscle chamber was switched to a formulation that supports myotube fusion and NMJ formation. MNs with fAD mutations (PSEN 1, APP) and WT MNs (control) and WT MNs isogenic to the APP mutation (iso WT) were plated into MN chambers approximately two days later, followed by functional testing on days 11, 17, and 23 from the time of the MN plating. NMJs from the PSEN1 MNs indicated a significant deficit in fidelity compared to the WT control at all testing frequencies on all testing days (days 11, 17, and 23) (Figure 5 ). NMJs from the APP MNs showed a significant deficit compared to the isogenic control on day 17 at all testing frequencies (Figure 5B ), while on day 23, the deficit did not indicate statistical significance. NMJs formed with PSEN1 MNs showed increased fatigue index compared to the WT control at 2 Hz stimulation frequency at all testing days with a significant increase on testing days 11 and 17 (Figure 6B ). However, NMJs formed with APP MNs had increased fatigue index at all testing days compared to the isogenic control, but the deficits did not have statistical significance (Figure 6B ). The average number of NMJs formed in systems with fAD MNs showed no significant difference compared to controls across all testing days (Figure 6Ci ). However, NMJ stability data indicates that systems with the PSEN1 MNs had a significant deficit in the maintenance of NMJs at testing day 17 compared to the WT control (Figure 6Cii ), while on other testing days, the reduction of NMJ stability did not have statistical significance. FIGURE 5. Open in a new tab Fidelity analysis of NMJs consisting of iPSC‐AD‐MNs and WT SKM, recorded through indirect (MN‐side) stimulation. (A) Example traces at testing day 17 at 0.33 Hz, 0.5 and 1 Hz stimulation frequency; (B) Quantification of NMJ fidelity at the testing days of 11, 17, 23 for frequencies of: (i) 0.33 Hz, (ii) 0.5 Hz, (iii) 1 Hz, (iv) 2 Hz; N = 3 (Number of times experiment was repeated); Data collected from more than 3 independent biological replicates per condition; One‐way ANOVA, Sidak Multiple comparison ( p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***)). FIGURE 6. Open in a new tab Fatigue Index analysis of NMJs consisting of iPSC‐AD‐MNs and WT SKM, recorded through indirect (MN‐side) stimulation. (A) Example of indirect traces at testing day 17 at 2 Hz stimulation frequency; (B) Quantification of NMJ fatigue index at the testing days of 11, 17, 23 for frequencies of: i) 2 Hz; (WT HMN, PSEN1 HMN, Iso WT HMN, and Iso APP HMN), N = 3; One‐way ANOVA, Sidak Multiple comparison. ( p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***)). (C) (i) Average number of NMJ per system at Day 11, 17, and 23; (ii) % Stability at Day 11, 17 ad 23, N = 3 (Number of times experiment was repeated); Data collected from more than 3 independent biological replicates per condition; One‐way ANOVA, Sidak Multiple comparison. ( p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***)). As a control, the WT SKM in the NMJ chambers was electrically stimulated directly in the muscle chamber (direct stimulation) at 0.33, 0.5, 1, and 2 Hz frequencies. The WT SKM cultured with WT MNs, PSEN1 MNs, Isogenic Control MNs and APP MNs showed no deficit in direct fidelity under each direct electrical stimulation frequency protocol (Figure 7A, B ) and for fatigue index (Figure 7C ). After functional testing was conducted, chambers were fixed in PFA and stained for MN (SMI312) and SKM specific markers (MHC) to visualize NMJs formed between WTA SKM and iPSC‐fAD‐MNs. This was performed as added evidence that activity data gathered in this study was from altered NMJ function (Figure 8 ). Overall, no morphological deficits were observed in NMJs formed between WTA SKM and iPSC‐fAD‐MNs. FIGURE 7. Open in a new tab Direct fidelity and fatigue index analysis of WT SKM that was used in the NMJ experiment with Ipsc‐AD‐MNs. A) Example of direct traces at testing day 17 at 1 Hz stimulation frequency; B) Quantification of direct fidelity at the testing days of 11, 17, and 23 for frequencies: (i) 0.33 Hz, (ii) 0.5 Hz, (iii) 1 Hz, (iv) 2 Hz; (C) Quantification of direct fatigue index at the testing days of 11, 17, 23 for 2 Hz stimulation frequency; N = 3 (Number of times experiment was repeated); Data collected from more than 3 independent biological replicates per condition; One‐way ANOVA, Sidak Multiple comparison ( p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***)). FIGURE 8. Open in a new tab NMJ formation and maintenance, iPSC‐AD‐MNs and WT SKM. Representative MHC, SMI312 and BTX immunocytochemistry images of NMJ chambers with WT SKM and fAD MNs at day 14, scale bar 50 µm. 3.3. Functional analysis of NMJ systems after the AD‐MNs were treated with galantamine and memantine Memantine was administrated to the MN chambers at the final concentration of 5 µM on day 13 from MN plating. NMJ functional measurements were conducted on Day 11 as a pre‐treatment baseline, and at 24 h, 72 h and 1 week for evaluating the effect of the drug (Figure S2 ). Similar to the previous experiments, NMJs formed with PSEN1 MNs indicated deficits in fidelity at all stimulation frequencies in comparison to NMJs formed with WT MNs at all testing days (Figure 2 ). No significant change in fidelity was observed in NMJs formed with PSEN1 MNs that were treated with memantine in comparison to the untreated PSEN1 group across all testing days, post baseline (Figure 2 ). A non‐significant decrease in fidelity was observed in NMJs formed with WT MNs that were treated with memantine in comparison to the untreated controls across all testing days, post baseline (Figure 2 ). During the drug treatment experiment, NMJs formed with PSEN1 MNs showed deficits in fatigue index compared to the NMJs formed with WT MNs across all testing days (Figure 3Ai ). No significant change in the fatigue index was observed in NMJs formed with PSEN1 MNs that were treated with memantine compared to the untreated PSEN1 group (Figure S3Ai ). A non‐significant deficit in fatigue index was observed in NMJ systems with WT MNs that were treated with memantine in comparison to untreated WT MN group post baseline testing days (Figure S3Ai ). No significant deficit was observed during the memantine treatment experiment in the number of NMJs formed between WT SKM and PSEN1 MNs in comparison to WT MNs, and memantine treatment did not induce any significant change for either WT or PSEN1 NMJs (Figure S3Bi ). Irregardless of the drug treatment, NMJs formed with PSEN1 MNs showed a deficit in stability during the baseline testing day, 24‐h post drug treatment and 72‐h post drug treatment in comparison to NMJs formed with WT MNs (Figure S3Bii ), but no significant difference was identified between treated versus untreated groups for both WT and PSEN1 conditions (Figure S3Bii ). As a control, direct stimulation of the SKM cultured in the NMJ chambers where fAD MNs were treated with memantine showed no deficit in fidelity (Figure S4 ). Galantamine was administered to either SKM or fAD MNs co‐cultured in the NMJ system at a clinically relevant concentration (10 µM). 40 This is because one of its targets, the acetyl choline receptor, unlike memantine can be found on the MNs and SKM. The NMJ experiments were conducted in a similar timeline as previously described in memantine treatment experiments. Administration of galantamine to PSEN1 MNs co‐cultured with WT SKM did not result in improvement in fidelity (Figure S5 ), fatigue index (Figure S6A ), NMJ number (Figure S6Bi ), and stability (Figure S6Bii ). Direct stimulation of WT SKM co‐cultured with PSEN1 MNs that were treated with galantamine showed no deficit in fidelity (Figure S7 ). Administration of galantamine to WT SKM co‐cultured with PSEN1 MNs showed no improvement in fidelity (Figure S8 ), fatigue index (Figure S9A ), NMJ number (Figure S9 Bi ), and stability (Figure S9 Bii ). As a control, direct stimulation of WT SKM treated with galantamine and co‐cultured with PSEN1 MNs showed no deficit in fidelity (Figure S10 ). 4. DISCUSSION In this study, MNs were differentiated from iPSCs harboring fAD mutations (PSEN1 A246E and APP K595N/M596L, Swedish mutation). The pathology of NMJs formed between fAD MNs with WT skeletal muscle were investigated by utilizing an established NMJ Bio‐MEMs functional assay. 17 , 21 , 22 , 39 , 41 Data gathered from the functional NMJ assay showed that MNs with PSEN1 and APP mutations were capable of forming NMJs with WT skeletal muscle at similar numbers compared to WT and isogenic control MNs (Figure 6Ci ). NMJs formed between APP MNs and WT skeletal muscle showed a significant deficit in indirect fidelity on the second testing day (day 17) (Figure 5Bi, ii ). NMJs with APP MNs indicated no deficit in fidelity on early (day 11) or late (day 23) testing days, nor did they show a deficit in fatigue index and functional NMJ stability compared to isogenic control MNs (Figures 5 and 6 ). In comparison, NMJs with PSEN1 MNs showed a deficit in fidelity at all tested days, and an increase in fatigue index on day 11 and day 17, as well as a significant deficit in NMJ stability at day 17 (Figures 5 and 6 ). These functional data indicate the NMJ deficits were induced by fAD mutations in MNs, while revealing the heterogeneity of the pathology between different mutations. Since the muscle utilized during these experiments was differentiated from the iPSCs of a healthy donor, any NMJ functional deficiencies observed in this study are attributed to fAD MNs. The functional integrity of the muscle in the NMJ chambers was confirmed through direct electrical stimulation protocols applied on the SKM chamber (Figure 7B ). The NMJ fidelity and fatigue index could be affected by MN excitability, insufficient supply and transport of acetylcholine/synaptic vesicles and mitochondria, ineffective release of the acetylcholine, and reuptake of choline which are important for the maintenance of activities at the axon terminal. 21 According to the current clamp data, APP MNs showed a deficiency in induced repetitive firing at DIV 19, which was near the second NMJ testing point at DIV 17, suggesting that reduced excitability could have contributed to the deficiency in NMJ fidelity at that time point (Figure 3 ). PSEN1 MNs were hypoexcitable (lower sodium/potassium currents ratio) at DIV 6, hyperexcitable (reduced resting membrane potential) at DIV 12, and hypoexcitable (lower sodium/potassium currents ratio) at DIV 19 (Figure 3 ). The dysregulated MN excitability may have contributed to the poor NMJ fidelity with PSEN1 MNs. One of the key enzymes responsible for the processing of APP into the Aβ, β‐secretase (BACE1), is also responsible for the modulation of total production and surface expression of voltage‐gated Na v 1 channels. 42 , 43 There is evidence that Aβ and the presence of pathogenic Aβ plaques positively regulate the expression of BACE1, which in turn can alter the excitability of the neurons through modulating surface expression of Na v 1 channels. 44 Since it is known that APP processing in both APP and PSEN1 mutations results in either an increase in total amyloid or more pathogenic variants of Aβ, it can be inferred that the excitability of neurons can be dysregulated through differential modulation of Na v 1channels by the increase of Aβ production over time. 38 Also, it cannot be ruled out that neurons with fAD mutations experience a shift in the population over time with hyperactive neurons dying, leaving only neurons that are less active remaining as is known from MN diseases such as ALS. 26 , 27 The Rab5 immunocytochemistry analysis indicated an increased number of Rab5‐positive enlarged early endosomes in fAD MNs compared to WT (Figure 4 ). In neurons, activation/deactivation of Rab5 GTPase is an essential controller of endocytosis at the axon terminal, and of subsequent retrograde transport of neurotropic factors to the soma, such as nerve growth factor and brain‐derived neurotropic factor. 45 , 46 , 47 Also, Rab5 plays a role in maintaining the uniform size of the synaptic vesicles, where activated Rab5 triggers the fusion of the synaptic vesicles with each other. 48 Additionally, during cell stress, inactivated Rab5 translocate to mitochondria, where it is activated by Alsin, and activated Rab5 can interact with factors that can protect mitochondria during stress. 49 , 50 Therefore, Rab5 dysregulation can cause dysfunctional retrograde axonal transport, dysregulation of the synaptic vesicle size, and aberrant mitochondrial stress protection. The altered Rab5 immunocytochemistry in PSEN1 and APP MNs strongly suggests that Rab5 dysregulation might contribute to the poor functional fidelity in fAD NMJ systems. Based on NMJ functional data, fAD mutations in MNs do not significantly affect the ability of the axons to innervate skeletal muscle. Only PSEN1 MNs indicated a significant deficit in NMJ stability at the second testing point on day 17 (Figure 6C ). Presumably, lowerγ‐secretase activity, a reported causal event for the development of tauopathy, and/or enlargement of early endosomes in PSEN1 mutations could possibly have contributed to poor maintenance of NMJs formed with PSEN1 MNs. 51 Memantine is an FDA approved NMDA receptor antagonist commonly administrated to patients with mild AD for its ability to alleviate cognitive symptoms and tolerability. 52 Overactivation of NMDA receptors in neurons can lead to excitotoxicity due to the increased Ca 2+ influx, and non‐competitive NMDA receptor antagonists such as memantine can alleviate excitotoxicity. 52 MNs do express the NMDA receptor and it has been reported that NMDA receptor modulation and excitotoxicity play a role in MN pathology of ALS. 53 However, memantine treatment of PSEN1 MNs in the NMJ system yielded no improvement on any functional parameters tested (Figures S2 , S3 and S4 ). Cholinesterase inhibitors such as galantamine are commonly prescribed to AD patients since there is a link between neurodegeneration and impaired cholinergic signaling, i.e. due to the loss of cholinergic neurons or the reduction of nicotinic acetylcholine receptors. 54 , 55 Inhibition of acetylcholine esterases allows prolonged action of acetylcholine at the synaptic cleft which can be beneficial during AD. 56 Additionally, galantamine can allosterically modify nicotinic acetylcholine receptors which improve their response to acetylcholine. 56 Due to these properties, galantamine was administered either to WT SKM or fAD MNs chamber in the NMJ system. Galantamine treatment of SKM or fAD MNs yielded no functional improvement in NMJ parameters (Figures S5–S10 ). Based on these results, it strongly suggests combinational therapeutics should be developed that can target cognitive and non‐cognitive symptoms of AD pathology. In this study, NMJ deficits were only investigated in a basic fAD NMJ model consisting of SKM and MNs. The addition of other cell types into the system, such as Schwan cells or astrocytes, could have a compensatory effect or exasperate the phenotype. Further studies are needed to evaluate the effect of additional cell types and whether Aβ can cause dysregulation of the MN excitability and NMJ functional deficit. Compounds that could improve the functional performance of fAD MNs could be screened in this NMJ model, which has only been available previously with animal models. The dual chamber system, which enables chemical and electrical isolation between skeletal muscle and MNs, would allow target‐specific drug testing. 21 In the future, the functional phenotypes of the SKM differentiated from fAD iPSCs could be evaluated in both the NMJ and a cantilever assay developed by our group. Since AD patients experience reduction in strength, the cantilever assay could help evaluate strength of fAD muscle and Aβ 42 has been found in the peripheral tissues of postmortem AD patients. 2 , 14 Lastly, due to the availability of the NMJ chamber system, the fAD iPSCs lines and differentiation protocols for sensory neurons and intrafusal fibers, a potential functional assay could be developed to investigate the pathology of the afferent pathways for AD. 53 , 57 , 58 5. CONCLUSION Utilizing a human neuromuscular platform, iPSC MNs lines carrying the PSEN1 A246E mutation and APP “Swedish” mutation were phenotypically analyzed. Patch clamp analysis revealed fAD MNs experienced dysregulation in excitability. Functional NMJ analysis demonstrated PNS motor deficits in fAD PNS independent of CNS. Drug treatment with the two drugs routinely used to treat AD did not show therapeutic effect on the PSEN1 mutant systems, suggesting different pathways would need to be targeted for PNS pathology and CNS pathology. Since the NMJ models have already been utilized to provide efficacy data for successful INDs for MG, CMT disease and ALS that were authorized for Phase II clinical trials, it makes evaluation of potential therapeutics for motor symptoms of AD straightforward. AUTHOR CONTRIBUTIONS Conceptualization : James J. Hickman, Xiufang Guo and Akhmetzada Kargazhanov. Investigation : Akhmetzada Kargazhanov, Romy Aiken, Gaurav Srivastava, Kenneth Hawkins, Rafael Lopez, Chase Miller, Will Bogen and Ahmad Nawaz. Methodology : James J. Hickman, Xiufang Guo and Christopher Long. Original data curation : Akhmetzada Kargazhanov. Review of data curation : Xiufang Guo, Dave Morgan, and James J. Hickman. Formal data analysis : Akhmetzada Kargazhanov and Xiufang Guo. Funding acquisition : James J. Hickman. Experimental guidance : Xiufang Guo, Dave Morgan and James J. Hickman. Project administration : Xiufang Guo, Christopher Long and James J. Hickman. Supervision : Xiufang Guo and James J. Hickman. Validation : Xiufang Guo, Dave Morgan, and James J. Hickman. Visualization : Akhmetzada Kargazhanov. Writing—original draft : Akhmetzada Kargazhanov. Writing—review and editing : Akhmetzada Kargazhanov, Xiufang Guo, Dave Morgan, and James J. Hickman. CONFLICT OF INTEREST STATEMENT James J. Hickman has ownership interest and is Chief Scientist and member of the Board of Directors in a company that may benefit financially as a result of the outcomes of the research or work reported in this publication. All other authors declare no financial interests. Author disclosures are available in the supporting information . Supporting information Supporting Information ALZ-22-e71281-s012.docx (23.7KB, docx) Supporting Information ALZ-22-e71281-s011.docx (367KB, docx) Supporting Information ALZ-22-e71281-s010.pdf (1.3MB, pdf) Supporting Information ALZ-22-e71281-s007.pdf (888.9KB, pdf) Supporting Information ALZ-22-e71281-s001.docx (562.5KB, docx) Supporting Information ALZ-22-e71281-s005.pdf (1.2MB, pdf) Supporting Information ALZ-22-e71281-s006.docx (523.9KB, docx) Supporting Information ALZ-22-e71281-s009.docx (534.7KB, docx) Supporting Information ALZ-22-e71281-s002.docx (1,008.9KB, docx) Supporting Information ALZ-22-e71281-s003.pdf (892.8KB, pdf) Supporting Information ALZ-22-e71281-s004.pdf (2.1MB, pdf) Supporting Information ALZ-22-e71281-s008.pdf (778KB, pdf) ACKNOWLEDGMENTS This project was supported by the Institute of Aging at the National Institutes of Health (grants number R01AG077651 and R44AG071386). REFERENCES 1. Aging NIo . Alzheimer's disease fact sheet. 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