Candidate CSPG4 mutations and induced pluripotent stem cell modeling implicate oligodendrocyte progenitor cell dysfunction in familial schizophrenia

Schizophrenia is highly heritable, yet its underlying pathophysiology remains largely unknown. Among the most well-replicated findings in neurobiological studies of schizophrenia are deficits in myelination and white matter integrity; however, direct etiological genetic and cellular evidence has thus far been lacking. Here, we implement a family-based approach for genetic discovery in schizophrenia combined with functional analysis using induced pluripotent stem cells (iPSCs). We observed familial segregation of two rare missense mutations in Chondroitin Sulfate Proteoglycan 4 (CSPG4) (c.391G > A [p.A131T], MAF 7.79 × 10−5 and c.2702T > G [p.V901G], MAF 2.51 × 10−3). The CSPG4A131T mutation was absent from the Swedish Schizophrenia Exome Sequencing Study (2536 cases, 2543 controls), while the CSPG4V901G mutation was nominally enriched in cases (11 cases vs. 3 controls, P = 0.026, OR 3.77, 95% CI 1.05–13.52). CSPG4/NG2 is a hallmark protein of oligodendrocyte progenitor cells (OPCs). iPSC-derived OPCs from CSPG4A131T mutation carriers exhibited abnormal post-translational processing (P = 0.029), subcellular localization of mutant NG2 (P = 0.007), as well as aberrant cellular morphology (P = 3.0 × 10−8), viability (P = 8.9 × 10−7), and myelination potential (P = 0.038). Moreover, transfection of healthy non-carrier sibling OPCs confirmed a pathogenic effect on cell survival of both the CSPG4A131T (P = 0.006) and CSPG4V901G (P = 3.4 × 10−4) mutations. Finally, in vivo diffusion tensor imaging of CSPG4A131T mutation carriers demonstrated a reduction of brain white matter integrity compared to unaffected sibling and matched general population controls (P = 2.2 × 10−5). Together, our findings provide a convergence of genetic and functional evidence to implicate OPC dysfunction as a candidate pathophysiological mechanism of familial schizophrenia

Modulation of Insulin/IGF Signalling to Improve Mammalian Healthspan

Research into ageing has revealed the malleability of the process and the importance of sex differences in modulating the response to longevity interventions. The importance of sex in mediating healthy ageing is evident in the brain, where sex is a major risk factor for some disorders. For example, women are twice as likely as men to suffer from Alzheimer’s Disease and men twice as likely as women to incur Parkinson’s Disease, two common and debilitating age-associated neurological disorders. My thesis underscores the importance of including both sexes when performing exploratory scientific studies where the effect of sex on a variable is unknown. Suppressed activity of the insulin/insulin-like growth factor signalling (IIS) pathway is a highly conserved and robust intervention that ameliorates the effects of ageing. While many invertebrate studies have dissected the role of IIS in ageing and have found tissue- and sex-specific effects, the role of different tissues and impact of sex in the longevity benefits of IIS reduction in mammals remain elusive. To address this gap, I first fully characterised male and female insulin receptor substrate 1 (Irs1) knockout (Irs1KO) mice. Then, I generated tissue-specific Irs1KO models targeting the major insulin-sensitive metabolic organs (liver, muscle, fat and brain) in male and female mice, and assessed the effects on body composition, energy expenditure and peripheral metabolism I found neuronal IRS1 deletion to be unique in triggering sex-specific health benefits in male mice. In the first section of the Thesis, I shed light on a mechanism by which reduced neuronal insulin signalling mediates mitochondrial phenotypes in Irs1KO mice, and I highlight the sex-specific nature of the neuronal mitochondrial stress response. IRS1 deletion is a robust method for lifespan extension and induction of metabolic health benefits. However, the effect of IRS1 deletion on cognitive function has not been assessed. Therefore, I asked whether IRS1 deletion reduced age-associated cognitive decline in young and old female and male mice. First, I found that IRS1 deletion did not lead to deficits in locomotor or exploratory behaviour, but it did lead to an age-associated reduction in anxiety. Next, I found no significant difference in the capacity of Irs1KO mice to perform a hippocampus-dependent spatial learning task and form long-term memory. Finally, I assessed short-term memory and found a significant enhancement in old Irs1KO mice, suggesting a reduction in age-associated cognitive decline. In the second section, I present data indicating the lack of sex-specific deficits in Irs1KO cognitive and motor function as well as a mitigation of age-dependent cognitive decline. Cognitive decline, a risk factor for dementia, is a major source of dependence in the elderly population, leading to increased societal and economic burden. I tested whether neuronal IRS1 deletion would be sufficient to delay or prevent cognitive decline in an ageing mouse model. I used a genetic tool to target IRS1 deletion to a widespread neuronal population. I therefore assessed whether the genetic tool itself induced phenotypes that could confound the effects of Irs1 deletion before moving on to study nKO mice. First, I characterised behavioural and metabolic parameters in male and female mice that expressed Cre under the rat-Synapsin I promoter (Syn1Cre). I found that Syn1Cre expression alone led to a sex-specific increase in anxiety and an inability to perform a spatial learning task in male Syn1Cre mice. Moreover, I found evidence of increased human growth hormone (hGH) expression in the brain due to the expression of a Cre-hGH transcript, leading to a negative feedback loop in the growth pathway resulting in a sex-specific reduction of body size in male Syn1Cre mice. Finally, I tested peripheral metabolism and found no significant difference in glucose tolerance, insulin sensitivity, energy expenditure or locomotor activity in male or female Syn1Cre mice. In the third section, I report that the Syn1Cre genetic model may be an appropriate tool for the study of neuronal control of peripheral metabolism, but not for behavioural studies. Therefore, I was unable to move forward with the characterisation of the neuron-specific IRS1 mutant

Sex-specific effects of Cre expression in Syn1Cre mice

Abstract The Cre-loxP system has been used to generate cell-type specific mutations in mice, allowing researchers to investigate the underlying biological mechanisms of disease. However, the Cre-recombinase alone can induce phenotypes that confound comparisons among genotypes if the appropriate Cre control is not included. In this study, we characterised behavioural, morphological and metabolic phenotypes of the pan-neuronal Syn1Cre line. We found that these mice possess intact neuromuscular parameters but have reduced exploratory activity and a male-specific increase in anxiety-like behaviour. Moreover, we observed a male-specific deficit in learning and long-term memory of Syn1Cre mice that could be a result of decreased visual acuity. Furthermore, we found that over-expression of human growth hormone (hGH) from Syn1Cre results in a male-specific reduction in body weight and femur length, potentially through decreased hepatic Igf1 expression. However, metabolic characteristics of Syn1Cre mice such as glucose metabolism, energy expenditure and feeding were unaffected by the presence of Syn1Cre. In conclusion, our data demonstrate that Syn1Cre expression has effects on behavioural and morphological traits. This finding highlights the importance of including the Cre control in all comparisons, while the male-specific effects on some phenotypes highlight the importance of including both sexes

Role of the Retinoblastoma protein, Rb, during adult neurogenesis in the olfactory bulb

Adult neural stem cells (aNSCs) are relatively quiescent populations that give rise to distinct neuronal subtypes throughout life, yet, at a very low rate and restricted differentiation potential. Thus, identifying the molecular mechanisms that control their cellular expansion is critical for regeneration after brain injury. Loss of the Retinoblastoma protein, Rb, leads to several defects in cell cycle as well as neuronal differentiation and migration during brain development. Here, we investigated the role of Rb during adult neurogenesis in the olfactory bulb (OB) by inducing its temporal deletion in aNSCs and progenitors. Loss of Rb was associated with increased proliferation of adult progenitors in the subventricular zone (SVZ) and the rostral migratory stream (RMS) but did not alter self-renewal of aNSCs or neuroblasts subsequent migration and terminal differentiation. Hence, one month after their birth, Rb-null neuroblasts were able to differentiate into distinct subtypes of GABAergic OB interneurons but were gradually lost after 3 months. Similarly, Rb controlled aNSCs/progenitors proliferation in vitro without affecting their differentiation capacity. This enhanced SVZ/OB neurogenesis associated with loss of Rb was only transient and negatively affected by increased apoptosis indicating a critical requirement for Rb in the long-term survival of adult-born OB interneurons

Candidate CSPG4 mutations and induced pluripotent stem cell modeling implicate oligodendrocyte progenitor cell dysfunction in familial schizophrenia

Schizophrenia is highly heritable, yet its underlying pathophysiology remains largely unknown. Among the most well-replicated findings in neurobiological studies of schizophrenia are deficits in myelination and white matter integrity; however, direct etiological genetic and cellular evidence has thus far been lacking. Here, we implement a family-based approach for genetic discovery in schizophrenia combined with functional analysis using induced pluripotent stem cells (iPSCs). We observed familial segregation of two rare missense mutations in Chondroitin Sulfate Proteoglycan 4 (CSPG4) (c.391G > A [p.A131T], MAF 7.79 × 10-5 and c.2702T > G [p.V901G], MAF 2.51 × 10-3). The CSPG4 A131T mutation was absent from the Swedish Schizophrenia Exome Sequencing Study (2536 cases, 2543 controls), while the CSPG4 V901G mutation was nominally enriched in cases (11 cases vs. 3 controls, P = 0.026, OR 3.77, 95% CI 1.05-13.52). CSPG4/NG2 is a hallmark protein of oligodendrocyte progenitor cells (OPCs). iPSC-derived OPCs from CSPG4 A131T mutation carriers exhibited abnormal post-translational processing (P = 0.029), subcellular localization of mutant NG2 (P = 0.007), as well as aberrant cellular morphology (P = 3.0 × 10-8), viability (P = 8.9 × 10-7), and myelination potential (P = 0.038). Moreover, transfection of healthy non-carrier sibling OPCs confirmed a pathogenic effect on cell survival of both the CSPG4 A131T (P = 0.006) and CSPG4 V901G (P = 3.4 × 10-4) mutations. Finally, in vivo diffusion tensor imaging of CSPG4 A131T mutation carriers demonstrated a reduction of brain white matter integrity compared to unaffected sibling and matched general population controls (P = 2.2 × 10-5). Together, our findings provide a convergence of genetic and functional evidence to implicate OPC dysfunction as a candidate pathophysiological mechanism of familial schizophreni