One of the prevailing hypotheses suggests schizophrenia as a neurodevelopmental disorder, involving dysfunction of dopaminergic and glutamatergic systems. Accumulating evidence suggests mitochondria as an additional pathological factor in schizophrenia. An attractive model to study processes related to neurodevelopment in schizophrenia is reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) and differentiating them into different neuronal lineages. iPSCs from three schizophrenia patients and from two controls were reprogrammed from hair follicle keratinocytes, because of their accessibility and common ectodermal origin with neurons. iPSCs were differentiated into Pax6(+)/Nestin(+) neural precursors and then further differentiated into β3-Tubulin(+)/tyrosine hydroxylase(+)/DAT(+) dopaminergic neurons. In addition, iPSCs were differentiated through embryonic bodies into β3-Tubulin(+)/Tbox brain1(+) glutamatergic neurons. Schizophrenia-derived dopaminergic cells showed severely impaired ability to differentiate, whereas glutamatergic cells were unable to maturate. Mitochondrial respiration and its sensitivity to dopamine-induced inhibition were impaired in schizophrenia-derived keratinocytes and iPSCs. Moreover, we observed dissipation of mitochondrial membrane potential (Δψm) and perturbations in mitochondrial network structure and connectivity in dopaminergic along the differentiation process and in glutamatergic cells. Our data unravel perturbations in neural differentiation and mitochondrial function, which may be interconnected, and of relevance to dysfunctional neurodevelopmental processes in schizophrenia.

Dysfunction of mitochondria, key players in various essential cell processes, has been repeatedly reported in schizophrenia (SZ). Recently, several studies have reported functional recovery and cellular viability following mitochondrial transplantation, mostly in ischemia experimental models. Here, we aimed to demonstrate beneficial effects of isolated active normal mitochondria (IAN-MIT) transfer in vitro and in vivo, using SZ-derived induced pluripotent stem cells (iPSCs) differentiating into glutamatergic neuron, as well as a rodent model of SZ. First, we show that IAN-MIT enter various cell types without manipulation. Next, we show that IAN-MIT transfer into SZ-derived lymphoblasts induces long-lasting improvement in various mitochondrial functions including cellular oxygen consumption and mitochondrial membrane potential (Δ ψ m). We also demonstrate improved differentiation of SZ-derived iPSCs into neurons, by increased expression of neuronal and glutamatergic markers β3-tubulin, synapsin1, and Tbr1 and by an activation of the glutamate-glutamine cycle. In the animal model, we show that intra-prefrontal cortex injection of IAN-MIT in adolescent rats exposed prenatally to a viral mimic prevents mitochondrial Δ ψ m and attentional deficit at adulthood. Our results provide evidence for a direct link between mitochondrial function and SZ-related deficits both in vitro and in vivo and suggest a therapeutic potential for IAN-MIT transfer in diseases with bioenergetic and neurodevelopmental abnormalities such as SZ.

Disease-specific induced pluripotent stem cells (iPSC) allow unprecedented experimental platforms for basic research as well as high-throughput screening. This may be particularly relevant for neuropsychiatric disorders, in which the affected neuronal cells are not accessible. Keratinocytes isolated from hair follicles are an ideal source of patients' cells for reprogramming, due to their non-invasive accessibility and their common neuroectodermal origin with neurons, which can be important for potential epigenetic memory. From a small number of plucked human hair follicles obtained from two healthy donors we reprogrammed keratinocytes to pluripotent iPSC. We further differentiated these hair follicle-derived iPSC to neural progenitors, forebrain neurons and functional dopaminergic neurons. This study shows that human hair follicle-derived iPSC can be differentiated into various neural lineages, suggesting this experimental system as a promising in vitro model to study normal and pathological neural developments, avoiding the invasiveness of commonly used skin biopsies.

Mitochondria, similar to living cells and organelles, have negative membrane potential and can therefore accumulate permeable lipophilic cations. Those cations which exhibit fluorescence activity after accumulation into energized systems are widely used to decipher changes in membrane potential by imaging techniques. Here we describe the use of the lipophilic cation 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine iodide (JC-1), which alters reversibly its color from green (J-monomer, at its low concentration in the cytosol) to red (J-aggregates, at its high concentration in active mitochondria) with increasing mitochondrial membrane potential (Δψm). We show that in addition to changes in Δψm, this specific dye can be used to follow alterations in mitochondrial distribution and mitochondrial network connectivity. We suggest that JC-1 is a preferable probe to compare between treatment groups, as the ratio of green to red fluorescence intensities is used for analysis. This ratio depends only on the mitochondrial membrane potential and not on other mitochondrial dependent or independent factors. We demonstrate various applications of JC-1 staining to study mitochondrial abnormalities in different cell types derived from schizophrenia patients and healthy subjects.