A Kalirin missense mutation enhances dendritic RhoA signaling and leads to regression of cortical dendritic arbors across development

Normally, dendritic size is established prior to adolescence and then remains relatively constant into adulthood due to a homeostatic balance between growth and retraction pathways. However, schizophrenia is characterized by accelerated reductions of cerebral cortex gray matter volume and onset of clinical symptoms during adolescence, with reductions in layer 3 pyramidal neuron dendritic length, complexity, and spine density identified in multiple cortical regions postmortem. Nogo receptor 1 (NGR1) activation of the GTPase RhoA is a major pathway restricting dendritic growth in the cerebral cortex. We show that the NGR1 pathway is stimulated by OMGp and requires the Rho guanine nucleotide exchange factor Kalirin-9 (KAL9). Using a genetically encoded RhoA sensor, we demonstrate that a naturally occurring missense mutation in Kalrn, KAL-PT, that was identified in a schizophrenia cohort, confers enhanced RhoA activitation in neuronal dendrites compared to wild-type KAL. In mice containing this missense mutation at the endogenous locus, there is an adolescent-onset reduction in dendritic length and complexity of layer 3 pyramidal neurons in the primary auditory cortex. Spine density per unit length of dendrite is unaffected. Early adult mice with these structural deficits exhibited impaired detection of short gap durations. These findings provide a neuropsychiatric model of disease capturing how a mild genetic vulnerability may interact with normal developmental processes such that pathology only emerges around adolescence. This interplay between genetic susceptibility and normal adolescent development, both of which possess inherent individual variability, may contribute to heterogeneity seen in phenotypes in human neuropsychiatric disease.

Schizophrenia (SZ) is a debilitating disease that affects ∼1% of the population (1). Clinical symptoms, such as auditory hallucinations and delusions, emerge during the second or third decade of life. Recent studies, however, have emphasized that it is impairments in cognitive and sensory processes underlying the clinical symptoms that are the greatest contributors to functional impairment and long-term disability (2–4). Specifically, auditory processing deficits have been consistently described at the neurophysiological level and include increased threshold for detecting differences between successive auditory stimuli and decreased amplitude of the mismatch negativity response to silent gaps, processes which require an intact of the auditory cortex (5, 6). Current therapeutics have limited efficacy for cognitive and sensory function impairments and confer substantial morbidity owing to undesired side effects, underscoring the need for therapeutics targeting underlying molecular mechanisms.Pyramidal cells (PCs) represent the most abundant neuronal type in the cerebral cortex (7), and their integrity is essential to the cognitive and sensory processes that are disrupted in SZ (5,8). Among the most consistent and highly replicated findings from human postmortem studies of SZ are reductions in dendrite length, branching, and spine density in layer 3 PCs (9). Interestingly, these deficits appear to be more reliably defined in layer 3, as layer 5 PCs have not been consistently shown to be impaired in postmortem studies of SZ (10). Because dendritic spines are the site of most excitatory synapses, much research to date has aimed to determine mechanisms of their reduction in SZ. However, total spine number is a function of both total dendrite length and spine density. Moreover, dendritic length and branching determine a PC’s receptive field (11, 12), help to segment computational compartments (13), and contribute substantially to how the received signals are integrated and transmitted to the cell body (14, 15). Thus, there is a compelling need to investigate dendritic length and branching alterations in SZ.One of the driving physiological requirements for dendritic morphogenesis is the flexibility for adjustment in development and in response to experience (16). Initial rapid growth establishes a nearly full-sized dendritic arbor prior to adolescence (17). While dendrites retain the physiological flexibility to undergo modest changes in branch points or angles to refine circuitry, the overall net arbor size remains relatively constant across adolescence and into adulthood due to a homeostatic balance between growth and retraction pathways (17, 18).Although rapid dendritic growth is complete prior to adolescence, this developmental epoch is a particularly active period of structural changes in the brain, leading to loss of cortical gray matter volume (19–22). In SZ, this reduction in gray matter volume is accelerated (23, 24), coincident with the onset of clinical symptoms (25, 26). The predominant component of cortical gray matter is neuropil, which comprises dendrites and axonal processes (27). It is possible that accelerated gray matter reductions during adolescence in SZ may be, in part, due to regression of dendritic architecture beginning during that time. It stands to reason, then, that a genetic susceptibility in a pathway involved in dendritic morphogenesis may be further exacerbated during the adolescent transition and lead to the onset of clinical symptoms of SZ.Among the genes found to influence dendritic morphogenesis (16, 28) is Kalrn. Of the multiple isoforms generated from the Kalrn gene through alternative splicing, the longer isoforms (KAL9 and KAL12) possess two guanine nucleotide exchange factor (GEF) domains, the second of which activates the GTPase RhoA. A missense mutation (rs143835330) coding for a proline to threonine amino acid change in the Kalrn gene (Kalrn-PT) was first identified in a resequencing analysis in individuals with SZ (29). The P→T change in Kalrn-PT is adjacent to the RhoA GEF domain in the KAL9 and KAL12 isoforms (29) and was shown to act as a modest gain of function for RhoA activity in a heterologous overexpression system (30). The activity of the first GEF domain, which activates Rac1, was shown to be unaltered by the Kalrn-PT mutation (30).Importantly, RhoA regulation of dendritic morphogenesis requires molecular precision. Although constitutively active RhoA reduces dendritic morphogenesis in rodent PCs (31, 32), expression of dominant negative (DN) RhoA fails to affect dendritic outgrowth (33). Thus, targeting specific pathways upstream of RhoA activation is necessary to rescue structural impairments. For example, p75 is a neurotrophin receptor which directly binds to Nogo receptor (NGR) and, in response to ligand binding, subsequently activates RhoA (34, 35). In cerebellar granule neurons, the KAL9 isoform of the Kalrn gene has been shown to directly bind to p75 and provide the GEF domain required for RhoA activation (34). The NGR1/p75/KAL9 pathway is known to restrict neurite outgrowth (34). Specifically disrupting NGR1-mediated signaling via Nogo neutralizing antibodies promotes neurite outgrowth and extension in vitro (34, 36), and in vivo knockdown of neuronal-specific Nogo-A leads to increases in both branching and total length in Layer 2/Layer 3 (L2/3) dendrites (37). Interestingly, increased levels of Nogo messenger RNA as well as elevated levels of KAL9 protein have been described in SZ (38, 39), suggesting enhanced activity of this pathway may contribute to the impairments in dendritic morphogenesis in disease. Although to date Nogo is the most well studied, numerous myelin-associated inhibitors (MAIs) have been identified as additional NGR1 ligands and similarly serve to limit neurite outgrowth (40). Interestingly, a highly potent MAI, oligodendrocyte-myelin glycoprotein (OMGp), increases in expression across adolescence (41).Thus, we hypothesized that Kalrn-PT would act as a RhoA gain of function in neurons and lead to adolescent-onset reductions in dendritic length and complexity.