Link between Dysregulated Protocadherin Pathway Activity and Schizophrenia Cortical Interneurons

What is Schizophrenia and the importance of induced pluripotent stem cells:

Schizophrenia is one of the most significant mental illness or disorder that affects the ability of an individual to think, feel and behave. It also interferes with the ability of individual to function in day to day lifestyle. Schizophrenia is characterised by both positive and negative symptom as well as cognitive deficits. It is believed to be heritable neurodevelopmental disease, which affects 1% of the adult population worldwide[4]. There are various anti-psychotic treatments and many new techniques have been developed to overcome this devastating disorder. However, cognitive and negative symptom do persist even after the treatment, which prevents the normal functioning of an individual [4]. Noh et al. demonstrated that Schizophrenia is not a monogenic disease instead there are many multiple risk gene and environmental factors which interacts with each other and produces clinical phenotype. Up until now, it has been bit hard and problematic to understand schizophrenia and it’s developmental mechanism due to inadequacy of the existing model, as animal models are less successful in producing negative and cognitive symptoms[3]. However, recent studies and research have discovered that induced pluripotent stem cells provide a possibility to study schizophrenia in live patient derived neurons[1]. These induced pluripotent stem cells (iPSCs) can be derived from various somatic cells, such as dermal fibroblasts, hair follicles, glutamatergic neurons and other neural subtypes etc. In addition, Shao et al. used this newly discovered approach, induced pluripotent stem cells to understand the mechanism of schizophrenia. To begin the study, 4 transcription factors integrating retroviral vector were transmitted into somatic cells to generate iPSCs[7]Then, cortical interneurons were generated using induced pluripotent stem cells which were derived from relatively small cohort, 14 healthy control and 14 subjects with schizophrenia.

Rationale for each different experimental approaches and the relative findings:

Initially, Shao and his colleagues, used footprint – free modified RNA method to generate iPSCs from healthy and schizophrenia fibroblasts. The generated iPSCs were then reprogrammed using RNA transfection and the modified iPSCs showed morphology which was similar to the human Pluripotent stem cells and they also expressed pluripotent stem cells markers. Later on, the iPSCs were differentiated using the modified protocol which resulted in the generation of homogenous population of Cortical Interneurons (cINs). The phenotype of this newly generated cINs was analysed using real – time PCR analysis and the results were compared with the undifferentiated iPSCs and induced glutamatergic neurons. After the analysis, it was found that cIN-specific genes were highly expressed in cIN groups compared to other cells or Non-cIN markers. This confounding results confirm the identity and homogeneity of the generated cIN cells. The generated cINs were grafted using electrophysiological analysis into the mouse brain to determine if the cINs generated form iPSCs were functional and authentic for the diseases modelling. Both the healthy control and schizophrenia cINs were then transduced with GFPs to allow optogenetic studies in order to view cortical slices of the mouse brain. As result, it was found that grafted cINs showed rapid desensitizing inward currents and sustained outward currents, which supported the expression of both voltage gated Na+ and K+ channels. However, there was no difference between the groups in those voltage dependent channels. In addition, Healthy control and schizophrenia cIN developed into functional cINs, whose neuronal properties are similar to those of endogenous interneurons. Furthermore, the synaptic properties of grafted human cINs were analysed to see if they integrate into adult brain circuitry and receive any synaptic inputs form the host cortical neurons. And as result, it was discovered that the human cINs form both the controls have functional postsynaptic machinery to receive excitatory synaptic inputs from host glutamatergic cortical neurons as well as the grafted cINs also have presynaptic machinery for GABA release but they inhibit host cortical neurons.

After the clarification of the authenticity and functionality of the cINs derived from the iPSCs, RNA-sequence analysis was performed to compare transcriptomes of healthy control cINs to schizophrenia cINs after 8 week differentiation. This was done to see if there is any schizophrenia cIN-specific abnormalities exists in gene expression during development. It was discovered that neuronal and cIN markers such as MAP2, DCX and GAD1,VGAT,SST, Lhx6 were highly expressed respectively. However, the expression of nonrelevant markers were relatively low and there was no difference in the expression of this markers between the both, healthy and schizophrenia samples. Later on, real time PCR was used to see if there is any difference in the selected gene, such as PCDH2 between healthy and diseased samples. There was no clear separation between the healthy and schizophrenia samples, but the selected gene PCDHA2 did exhibit significant difference in the expression of gene among the two samples. Due to the involvement of the genetic mutation in the schizophrenia, the size of samples were expanded to 14 healthy and 14 schizophrenia controls. Similar to last time, RNA-sequencing was performed and it was found that apart from PCDHA2, there are many other genes that are downregulated in schizophrenia. These genes include PCDHA3, PCDHA6 and PCDHA8. In addition, the real time PCR further confirmed significant decrease in the expression of PCDHA family members. It was also discovered that PCDHA2 eQTL SNPs were highly associated with schizophrenia and this is further supported by figure 8b of the paper[6]. This association of PCDHA2 supports the idea that there is potential role of schizophrenia risk loci in regulating the gene expression of PCDHA2. However, this aspect of the study did not achieve a statistical significance due to the smaller sample size However a final conclusion was drawn form the study. It was concluded that “schizophrenia risk locus may be related to the regulation of multiple protocadherin family members in addition to PCDHA members”[6]

Later in the study, Pcdha and Pcdhg knock out mice were used to determine the effect of protocadherin hypofunction on cIN development. Pcdhg knock out mouse showed severe lethal phenotype and it was discovered that Pcdhg is critical for the localisation of Pcdha and function. Where as Pcdha knock out mouse showed mild phenotype, however there was significant arborisation deficits in Pcdha knock out cINs upon analysis. This finding was further supported by decrease in neurite number from cell body as well as the total branch number and the neurite length were also reduced in the Pcdha knock out mouse. In addition, there was deficits in the inhibitory synapses formation of prefrontal cortex cINs in Pcdha knock out mice, but there was no significant difference in the excitatory synapses. Overall, it is clearly shown that protocadherin pathway is important for the normal development of cINs in the prefrontal cortex as well as in normal sensorimotor gating. Schizophrenia cINs were further tested to see if they also demonstrate similar phenotypic deficits, similar to those cIN deficits by protocadherin hypofunction in previous study. The healthy control and schizophrenia cINs were infected with a “limiting titer of lentivirus expressing GFP under the ubiquitin promoter”[6]. Analysis of these GFP cells showed that schizophrenia cINs have significant decrease in neurite number from soma, total branch numbers and neurite length compared to healthy cINs. Furthermore, Linear regression analysis revealed weak correlation which exist between the PCDHA family members and arborisation. This suggests that absence of a schizophrenia circuit environment leads to the intrinsic deficit in the formation of inhibitory synapses of schizophrenia cINs. Lastly, the final experiment was conducted to see whether the observes developmental deficits continue to be present in adult post-mortem brain. The analysis of 3rd layer of PV form both healthy and schizophrenia controls showed significant deficits in diseased post-mortem cINs which was indicated by decrease in neurite number from soma, total branch number and neurite length. There was also decrease in inhibitory synapses formation of post-mortem diseased cINs. The result were exactly similar to those of phenotype observed in developmental cINs. The only difference that was observed in adult post-mortem diseased cINs and not in phenotypic developmental cINs was that the excitatory synapses formation was relatively reduced in the post-mortem diseased cINs. This reduction in excitatory synapses can be associated with deficits in the glutamatergic neurons.

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Strength and weakness of the experimental approach and contribution to broader field:

The main purpose of study was well achieved as Shao and his colleagues were successful to find that the expression of protocadherins, a family of cell surface proteins which are significantly downregulated in schizophrenia cINs during development. In addition, they also discovered that altered expression of this gene led to disease-relevant phenotypes in knockout mice as well as in human schizophrenia cINs. The conducted experiment also shows the importance of using disease-relevant and homogenous population of cell in order to understand the effect of risk genotypes on both healthy and diseased controls. The study was conducted using human samples, more specifically using neurons that are affected in schizophrenia disease. Also, the study used live tissues except the last approach which used post-mortem brain slices and tissues. In addition, for better result the experiment was repeated and also there was increase in sample size form 8 individuals to 14 individuals. Also, the techniques that were used such as real time PCR which targeted protocadherin genes and other family members of protocadherin genes. However, the sample size chosen for this experiment could have been improve by choosing larger cohort of individual. A larger sample size also provides greater chance to work with more line and achieve more adequate statistical significance which was not possible to achieve using smaller sample size. It was also suggested that using large sample size, the common and rare variants of the schizophrenia disease may converge providing insights into cellular and molecular function of common variants[5]. This insight could results into identification and production of new therapeutic targets for such neuropsychiatric disorder[5]. In addition, it would have been much better to use same or much similar mouse model for the two different approaches, morphological changes in neurons/synapses and other for behaviour. In the study two different mouse models were used. This could have contributed to discrepancies of result, as each has different body structure as well as different morphology.

Furthermore, the study has contributed to the broader field of research and future by introducing new techniques. The study demonstrates the power of using homogenous and functional populations of specific neuronal subtype that are known to be affected in schizophrenia to probe the pathogenesis of schizophrenia[2]. Not only schizophrenia, but this new method of using homogenous populations of cells can be used to study any other developmental disease mechanisms as well as this techniques may provide a pathway for effective therapies or preventions in the future which can prevent the occurrence of such diseases. It was also discovered that PKC inhibitor reverts the schizophrenia cINs back in to normal phenotype. However, the mechanism is unclear but it is suggested that they are other molecules or mechanisms are involved which allows the reversion of the cINs, providing a future direction of research.

There are many questions and decisions that are left unanswered. The very first questions that can be raised is based on the validity of control. For this study, Shao et al. selected Caucasian males to eliminate gender and ethnicity variations. However, apart from these two factors what are other factors that could be introduced or eliminated to obtain valid statistical results? In addition, the last study conducted using post-mortem brain slices also possessed limitation and challenges. The results that were obtained from the study supported the link between schizophrenia and dysregulation in protocadherin pathway. However, it was not known if those model has any other age related disease or any prior known developmental disease mechanisms. In addition, Clozapine was used as first line treatment without gaining any knowledge of the prior history of the individuals. If these individuals selected for the study have been exposed to any different type of drugs previously, then there are chances that the exposure of drug in previous years could have affected the gene expression or neuron structure. These change in gene expression or neuron structure can be interpreted as a disease related phenotype.

In summary, The overall aim of the research was achieved, that there is correlation between protocadherin and schizophrenia disease, however again the exact mechanism is bit unclear and require further research. In addition, Shao et al. were able to identify the genes of PCDHA family that were downregulates in the schizophrenia cINs as well as they were also able to identify the affected pathway in the diseased individual. So, after all the discovery, findings and creating a direction for the future research, Shao et al. have immensely contributed to the broader field of research and the field of developmental disease mechanisms.

References:

1. Brennand, K.J., Gage, F.H., 2011. Concise Review: the promise of human induced pluripotent stem cell – based studies of schizophrenia. Stem cells 29 (12), 371, 1915 – 1922.
2. Jacobs, B.M., 2015. Concise Review: A dangerous method? The use of induced Pluripotent stem cells as a model for schizophrenia. Schizophrenia Research 168, 563 – 568.
3. Jones, C.A., Watson, D.J.G., Fone K.C.F., 2011. Animal models of schizophrenia. Br. J. Pharmacol. 164 (4), 1162 – 1194.
4. Noh, H., Shao Z., Coyle J.T., Chung S., Modelling schizophrenia pathogenesis using patient – derived induced pluripotent stem cells (iPSCs), 1863 (9): 2382 – 2387.
5. Rajarajan, P., Flaherty, E., Akbarian S., Brennand, K.J., Concise Review: CRISPR – based functional evaluation of schizophrenia risk variants. Schizophrenia Research, https://doi.org/10.1016/j.schres.2019.06.017
6. Shao Z., et al., 2019, Dysregulated protocadherin – pathway activity as an intrinsic defect in induced pluripotent stem cell – derived cortical interneurons from subjects with schizophrenia. Nature neuroscience, (22), 229 – 242.
7. Takahashi, K., Yamanaka, s., 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblasts cultures by defined factors. Cell 126(4), 663 – 676.