Clinical Studies
Biological mechanisms of neuropsychiatric disorders can be studied in clinical populations using multiple lines of inquiry. Structural brain imaging can reveal changes in brain region volume while functional imaging can demonstrate altered functioning of brain regions, both of which may originate in early developmental processes, particularly when found in patients presenting early in the course of illness or young subjects at high risk for psychiatric disorders. More specific mechanisms relevant to stem cell biology may be examined in the post mortem brain as well as genetic analysis of patients. Converging evidence from populations of patients with autism, schizophrenia and affective disorders suggests that stem cell biology is implicated in neuropsychiatric etiology and pathophysiology.

Autism
The brain structure of patients with autism spectrum disorders (ASD) including autism, Asperger's Disorder (AD) and pervasive development disorder not otherwise specified (PDD-NOS) has been found to be abnormal using several different approaches. It is now widely believed that at least some of the deficits are present very early in life and that abnormal embryonic brain development may be a contributor to later structural deficits.
The head circumference of some patients with autism has long been known to be larger (Davidovitch et al., 1996; Woodhouse et al., 1996; Fombonne et al., 1999; Miles et al., 2000) because of abnormal acceleration in growth in early infancy (Courchesne et al., 2003). While no studies have been published on the neuroanatomy of high-risk individuals before a diagnosis of autism is made, retrospective data have shown that children with macrocephaly and autism do not have increased head circumference at birth (Lainhart et al., 1997; Courchesne et al., 2003; Dementieva et al., 2005; Redcay and Courchesne, 2005) but begin to show larger head measures at about 4 months of age (Gillberg and de Souza, 2002; Courchesne et al., 2005; Redcay and Courchesne, 2005). These findings suggest that autism may be underlain by either problems in early postnatal life and/or processes of embryonic development on which these postnatal events depend. Structural imaging of individuals with autism has shown that differences in brain volume, including both white and gray matter, diminish after the age of 5 years (Hazlett et al., 2005), although some studies have reported increases in gray matter volume in adolescents and adults with ASD (Lotspeich et al., 2004; Palmen et al., 2005; Hazlett et al., 2006), particularly in PFC (Mitchell et al., 2009).
Although there is no clear mechanism accounting for the dysregulation in the trajectory of brain growth in ASD, one hypothesis stipulates that it is the consequence of altered regulation of neural stem cell proliferation or differentiation arising before birth (Vaccarino et al., 2009). Further evidence that early embryonic developmental events are implicated in the pathophysiology of autism comes from post mortem studies demonstrating a fundamental change in cortical structure. Patients with autism were shown to have an increased packing density of mini-columns, which are vertical (radial) assemblies of neurons thought to be anatomically and functionally interconnected (Casanova et al., 2003, 2006).
Several underlying mechanisms could explain macrocephaly and minicolumn pathology, all based on altered embryonic cortical development (Figure 3). The first is an increase in the number of radial units in the embryonic cerebral cortex, which in turn is thought to depend upon an increase in the number of “founder” NSCs in the cortical primordium (Rakic, 1995) (Figure 3). This mechanism is supported by the occurrence of mutations in pten, a gene that regulates embryonic stem cell proliferation (Eng, 2003) in a small number of autistic patients with macrocephaly (Butler et al., 2005). Abnormal expression of this gene in NSCs would likely result in an intrinsic alteration of stem cells. Interestingly, an animal model of pten mutations shows increased brain size and social deficits (Kwon et al., 2006), although this mutation was in differentiated neurons, not intrinsically affecting NSCs. In Fragile X syndrome, which frequently presents with symptoms of autism, fetal NSCs have been shown to differentiate into neurons at greater rates (Castren et al., 2005) and to misexpress multiple genes involved in proliferation and differentiation (Bhattacharyya et al., 2008). Mutant embryonic NSCs isolated from mice lacking the fragile X mental retardation protein (FMRP) due to a deletion in the fmr1 gene differentiate in greater numbers into immature neurons (Castren et al., 2005). These findings are similar to those obtained in Drosophila germline stem cells lacking an ortholog of the fmr1 gene (Yang et al., 2009). Thus, the pten and fmr1 mouse models of ASD support the hypothesis that an intrinsic abnormality in NSC is responsible for features of these disorders. Two members of the TF-II family of transcription factors involved in Williams syndrome, another disorder with abnormal social behavior, have been shown in mice to regulate specific gene targets that may be involved in embryonic stem cell differentiation (Makeyev and Bayarsaihan, 2009).
Figure 3  Two neuronal cell types, excitatory (blue) and inhibitory (red), contribute to minicolumn structure and functional balance in cortex. Autism associated genes play roles in the processes underlying the generation and migration of these cortical neurons. The disruption of these genes may determine some cortical abnormalities in autism. GABA interneurons surround each minicolumn, and some genetic evidence and post mortem data suggest a GABAergic abnormality in ASD (Fatemi et al., 2002). Multiple genes involved in the development and function of the GABAergic system, including dlx5, have been involved in Rett syndrome, a developmental abnormality with autistic features (Horike et al., 2005); furthermore, alterations in the development of GABAergic neuron circuitry have been found in mice lacking the Methyl-CpG binding protein 2 (MeCP2) gene, whose mutations are responsible for Rett syndrome (Medrihan et al., 2008; Zhang et al., 2010). Lastly, a large number of synaptic-related genes have been implicated in small subsets of patients with ASDs (in total, accounting for probably less than 3–4% of the cases) (Buxbaum, 2009; Radyushkin et al., 2009). Thus, it appears that abnormalities in both the early-determined size and scaffolding of the cerebral cortex and later developing synaptic connections may play a role in individual cases of autism.

Schizophrenia
In schizophrenia, retrospective studies have suggested that head circumference is decreased at birth and developmental delays are present in early childhood, both of which implicate prenatal and early postnatal alterations in forebrain development (Cannon et al., 2002). The occurrence of prodromal symptoms in the majority of cases (Hafner et al., 1994), as well as the presence both prior to and after onset of illness of neuropsychological deficits (Crespo-Facorro et al., 2007) also implicates disruption of forebrain development. Neuropsychological dysfunction implicates the PFC in patients with schizophrenia, the cortical region that has expanded most extensively in mammalian evolution and that has been suggested to rely on key evolutionary developments in stem cell function (Martinez-Cerdeno et al., 2006).
Altered hippocampal development has also been implicated in the pathogenesis of schizophrenia (Kobayashi, 2009). Total brain gray matter and hippocampal volumes are reduced at the time of first episode in schizophrenia (Ohnuma et al., 1997; Gur et al., 1999) and at times later in the disease course, volume reductions have been observed in PFC, hippocampus and temporal lobe (Shenton et al., 2001). Longitudinal studies with structural imaging show that both the PFC and the medial temporal lobe become progressively smaller during the period when psychosis develops and that dorsal prefrontal regions experience even more loss as illness progresses, consistent with a neurodegenerative process (Pantelis et al., 2007). Post mortem studies of the cerebral cortex of patients with schizophrenia have shown reductions in neuropil (Selemon et al., 1998), as well as in GABAergic cells expressing PV and reelin (Guidotti et al., 2000; Fatemi et al., 2001; Hashimoto et al., 2008). Post mortem analysis of the hippocampus also shows evidence for an abnormal GABAergic system and suggests that important signaling pathways are altered, including WNT and TGFbeta (Benes et al., 1998, 2007; Todtenkopf and Benes, 1998; Arnold et al., 2005). While these post mortem findings may reflect changes in the adult brain due to disease progression and medication effects, alterations in cortical neurodevelopmental processes could also create vulnerabilities that develop later in life.
Genes that are implicated in stem cell regulation, including neuregulin, disc-1, wnt related genes, bdnf and fgfr1, have been associated with schizophrenia in genetic association and post mortem studies, suggesting NSCs dysregulation in at least some cases. Mouse models lacking fgfr1 embryonically have smaller hippocampi and cortical interneuron deficits similar to those in patients with psychosis (Ohkubo et al., 2004). Similarly, deficient bdnf, neuregulin and disc-1 genes in mice mimic various aspects of the disorder (Ayhan et al., 2009; Brandon et al., 2009; Meyer and Morris, 2009). Conversely, mutations found in some patients with schizophrenia have significant effects on cortical stem cell development. Mice lacking the genes within the 22q11 mutation (velocardiofacial syndrome, a known chromosomal abnormality predisposing to schizophrenia) have abnormal neurogenesis, specifically affecting upper cortical layers (Meechan et al., 2009). As in autism, patients with schizophrenia are likely heterogenous, with pathology in some determined by a component of neuronal functioning that arises postnatally and in others, determined by earlier disruptions of patterning and neurogenesis.

Affective disorders
Patients with major depression have been found to have smaller hippocampi (Videbech and Ravnkilde, 2004) and smaller anterior cingulate cortical regions (Caetano et al., 2006). Connectivity and functioning of prefrontal and cingulate cortices have also been shown to be abnormal in individuals with major depression (Drevets, 2000; Grimm et al., 2008; Vasic et al., 2009). A recent longitudinal study of people at high familial risk for developing depression show thinning of the cerebral cortex, particularly in right-sided frontal and parietal areas (Peterson et al., 2009). This thinning was present even in children and adolescents prior to the onset of any mood episode, suggesting that structural changes that result from abnormal early cortical pruning during infancy or adolescence may be a predisposing factor in depression. In patients with bipolar disorder with psychotic symptoms, neuroanatomical and neuropsychological findings are similar to those found in patients with schizophrenia (Murray et al., 2004). In non-psychotic bipolar disorder, patients presenting at the first episode of illness have shown decreased volume in prefrontal and temporal cortex (Hirayasu et al., 1999; Strakowski et al., 1999; Kasai et al., 2003). Bipolar disease progression is associated with ventricular enlargement and stronger reductions of the PFC which correlate with impaired functioning during manic episodes (Bearden et al., 2001; Blumberg et al., 2003).
Post mortem studies have demonstrated that prefrontal cortical regions have abnormal densities of pyramidal, GABAergic and glial cells in patients with depression. Medium to large pyramidal neurons appear to be lower in density and smaller in soma size (Rajkowska et al., 1999, 2005; Law and Harrison, 2003) while smaller neurons and those in layers III, V may be increased in density (Chana et al., 2003; Rajkowska, 2003). Glial cell density in patients with depression appears significantly reduced (Ongur et al., 1998; Rajkowska et al., 1999; Cotter et al., 2001, 2002; Uranova et al., 2004). These findings suggest that abnormal prenatal development of stem/progenitor cells that generate the neurons populating different cortical layers and abnormal development of glial elements may account for structural brain abnormalities in depression.
Post mortem work has also examined the levels of signaling factors in patients with depression. In frontal cortical regions, FGF ligand and receptor levels as well as BDNF receptor levels were shown to be altered in patients with depression. Findings generally show reduced expression (Evans et al., 2004; Sibille et al., 2004) but some point to upregulated receptor levels (Tochigi et al., 2008). Altered gene expression in the mature cortex may suggest a role for FGF and other important mediators of neuronal growth in mood disorders in the adult brain. However, they may also reflect a genetic defect that may have contributed to an earlier altered developmental trajectory. Parallel findings in model animal systems (Chen et al., 2008) and patients with mood disorders (Rajkowska et al., 2001; Valentine and Sanacora, 2009) suggest a role for FGF in both embryonic and mature brain functions (Figure 4).
Figure 4  Evidence from model animal systems on fibroblast growth factor, cortical anatomy and functioning (top of each frame) converges with results from clinical studies, cortical anatomy and functioning (bottom of each frame) demonstrating how FGF may have a critical role in affective disorder psychopathology. FGF signaling contributes to normal cortical thickness and youth at risk for depression show early deficits in cortical thickness (A). The density of neurons in prefrontal cortex is reduced in both mice lacking FGF receptors and patients with bipolar disorder (B). A new target for antidepressant treatment is glial functioning in which FGF signaling plays a significant role (C). FGF receptor 1 plays a role in dentate gyrus adult neurogenesis in mice and, in patients with depression, antidepressant treatment has been correlated with increased dentate gyrus proliferation (D).