Human induced pluripotent stem cell line (ONHi001-A) generated from a patient with infantile neuroaxonal dystrophy having PLA2G6 c.517C > T (p.Q173X) and c.1634A > G (p.K545R) compound heterozygous mutations.

Infantile neuroaxonal dystrophy (INAD) is a rare neurodegenerative disease caused mainly by homozygous or compound heterozygous mutations in the PLA2G6 gene. We generated a human induced pluripotent stem cell (hiPSC) line (ONHi001-A) using fibroblasts derived from a patient with INAD. The patient exhibited c.517C > T (p.Q173X) and c.1634A > G (p.K545R) compound heterozygous mutations in the PLA2G6 gene. This hiPSC line may be useful for studying the pathogenic mechanism underlying INAD.

Postsynaptic structure formation of human iPS cell-derived neurons takes longer than presynaptic formation during neural differentiation in vitro.

The generation of mature synaptic structures using neurons differentiated from human-induced pluripotent stem cells (hiPSC-neurons) is expected to be applied to physiological studies of synapses in human cells and to pathological studies of diseases that cause abnormal synaptic function. Although it has been reported that synapses themselves change from an immature to a mature state as neurons mature, there are few reports that clearly show when and how human stem cell-derived neurons change to mature synaptic structures. This study was designed to elucidate the synapse formation process of hiPSC-neurons. We propagated hiPSC-derived neural progenitor cells (hiPSC-NPCs) that expressed localized markers of the ventral hindbrain as neurospheres by dual SMAD inhibition and then differentiated them into hiPSC-neurons in vitro. After 49 days of in vitro differentiation, hiPSC-neurons significantly expressed pre- and postsynaptic markers at both the transcript and protein levels. However, the expression of postsynaptic markers was lower than in normal human or normal rat brain tissues, and immunostaining analysis showed that it was relatively modest and was lower than that of presynaptic markers and that its localization in synaptic structures was insufficient. Neurophysiological analysis using a microelectrode array also revealed that no synaptic activity was generated on hiPSC-neurons at 49 days of differentiation. Analysis of subtype markers by immunostaining revealed that most hiPSC-neurons expressed vesicular glutamate transporter 2 (VGLUT2). The presence or absence of NGF, which is required for the survival of cholinergic neurons, had no effect on their cell fractionation. These results suggest that during the synaptogenesis of hiPSC-neurons, the formation of presynaptic structures is not the only requirement for the formation of postsynaptic structures and that the mRNA expression of postsynaptic markers does not correlate with the formation of their mature structures. Technically, we also confirmed a certain level of robustness and reproducibility of our neuronal differentiation method in a multicenter setting, which will be helpful for future research. Synapse formation with mature postsynaptic structures will remain an interesting issue for stem cell-derived neurons, and the present method can be used to obtain early and stable quality neuronal cultures from hiPSC-NPCs.

Older-onset levodopa-responsive parkinsonism with normal DAT-SPECT and pterin hypometabolism.

Pterin species participate in dopamine biosynthesis, and abnormal pteridine metabolism contributes to reduced dopamine. GTP cyclohydrolase 1 (GCH-1) deficiency, which triggers pteridine hypometabolism and normally develops in childhood, can mediate an adult-onset decrease in levodopa production and dopa-responsive dystonia (DRD), with normal dopamine transporter single-photon emission computed tomography (DAT-SPECT). A recent study described normal DAT-SPECT in adult-onset cases with GCH-1 mutations, clinically diagnosed with Parkinson's disease, which raises the possibility that the abnormal metabolism of pteridine may be a differential diagnosis for adult-onset parkinsonism. We report an older patient with levodopa-responsive parkinsonism with normal DAT-SPECT, or scans without evidence of dopamine deficit (SWEDD), whose biochemical analysis showed pterin hypometabolism, which occurs in GCH-1-deficient DRD. Surprisingly, this patient presented no dystonia or GCH-1 gene mutation or deletion. This case suggests that low pterin metabolism should be considered in older-onset levodopa-responsive parkinsonism with normal DAT-SPECT, even without GCH-1 mutations or deletions.

Alpha-synuclein dynamics in induced pluripotent stem cell-derived dopaminergic neurons from a Parkinson's disease patient (PARK4) with SNCA triplication.

Parkinson's disease (PD) is a neurodegenerative disorder caused by the selective loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). Lewy bodies (LBs), another histological hallmark of PD, are observed in patients with familial or sporadic PD. The therapeutic potential of reducing the accumulation of α-synuclein, a major LB component, has been investigated, but it remains unknown whether the formation of LBs results in the loss of DA neurons. PARK4 patients exhibit multiplication of the α-synuclein gene (SNCA) without any pathological mutations, but their symptoms develop relatively early. Therefore, study of PARK4 might help elucidate the mechanism of α-synuclein aggregation. In this study, we investigated the dynamics of α-synuclein during the early stage of immature DA neurons, which were differentiated from human-induced pluripotent stem cells (hiPSCs) derived from either a PARK4 patient with SNCA triplication or a healthy donor. We observed increased α-synuclein accumulation in PARK4 hiPSC-derived DA neurons relative to those derived from healthy donor hiPSCs. Interestingly, α-synuclein accumulation disappeared over time in the PARK4 patient-derived DA neurons. Moreover, an SNCA-specific antisense oligonucleotide could reduce α-synuclein levels during the accumulation stage. These observations may help reveal the mechanisms that regulate α-synuclein levels, which may consequently be useful in the development of new therapies for patients with sporadic or familial PD.