Development of the central nervous system is an incredible process that relies on multiple extracellular signaling cues and complex intracellular interactions. Approximately 1500 genes are associated with neurodevelopmental disorders, many of which are linked to a specific biochemical signaling cascade known as Extracellular-Signal Regulated Kinase (ERK1/2). Clearly defined mutations in regulators of the ERK1/2 pathway cause syndromes known as the RASopathies. Symptoms include intellectual disability, developmental delay, cranio-facial and cardiac deficits. Treatments for RASopathies are limited due to an in complete understanding of ERK1/2’s role in brain development. Individuals with Neurofibromatosis Type and Noonan Syndrome, the two most common RASopathies, exhibit aberrant functional and white matter organization in non-invasive imaging studies, however, the contributions of neuronal versus oligodendrocyte deficits to this phenotype are not fully understood. To define the cellular functions of ERK1/2 in motor circuit formation, this body of work focuses on two long-range projection neuron subtypes defined by their neurotransmitter. With genetic mouse models, pathological ERK1/2 in glutamatergic neurons reduces axonal outgrowth, resulting in deficits in activity dependent gene expression and the ability to learn a motor skill task. Restricting pathological ERK1/2 within cortical layer V recapitulates these wiring deficits but not the behavioral learning phenotype. Moreover, it is uncovered that pathological ERK1/2 results in compartmentalized expression pattern of phosphorylated ERK1/2. It is not clear whether ERK1/2 functions are similar in cholinergic neuron populations that mediate attention, memory, and motor control. Basal forebrain cholinergic neuron development relies heavily on NGF-TrKA neurotrophic signaling known to activate ERK1/2. Yet the function of ERK1/2 during cholinergic neuronal specification and differentiation is poorly understood. By selectively deleting ERK1/2 in cholinergic neurons, ERK1/2 is required for activity-dependent maturation of neuromuscular junctions in juvenile mice, but not the establishment of lower motor neuron number. Moreover, ERK1/2 is not required for specification of choline acetyltransferase expressing basal forebrain cholinergic neurons by 14 days of age. However, ERK1/2 may be necessary for BFCN maturation by adulthood. Collectively, these data indicate that glutamatergic neuron-autonomous decreases in long-range axonal outgrowth and modest effects on later stages of cholinergic neuron maintenance may be important aspects of neuropathogenesis in RASopathies.

The process of brain development is magnificently complex, requiring the coordination of millions of cells and thousands of genes across space and time. It is therefore unsurprising that brain development is frequently disrupted. Numerous genetic mutations underlying altered neurodevelopment have been identified and aligned with behavioral changes. However, the cellular mechanisms linking genetics with behavior are incompletely understood. The goal of my research is to understand how intracellular kinase signaling contributes to the development of ventrally derived glia and neurons. Of particular interest are GABAergic interneurons in the cerebral cortex, as GABAergic disruption is observed in multiple neurodevelopmental disorders including epilepsy, schizophrenia, and autism spectrum disorders. In addition, I investigated how kinase signaling influences the number and distribution of ventral born oligodendrocyte lineage cells to gain insight into white matter abnormalities observed in developmental disorders. This work primarily investigates the mitogen associated protein kinase (MAPK) signaling cascade, which is ubiquitously expressed but is particularly important for brain development. Hyperactive MAPK signaling causes RASopathies, a group of neurodevelopmental disorders where affected individuals often exhibit learning disability. MAPK haploinsufficiency, such as in 16p11.2 deletion syndrome, also results in intellectual disability. In both cases, the cells driving cognitive dysfunction are unknown. Using genetically modified mouse models, I found that hyperactivation of MAPK signaling disrupts a subtype of GABAergic neurons that express parvalbumin, though the same cells are resilient to MAPK deletion. In contrast, somatostatin expressing neurons require MAPK for normal development but are less responsive to hyperactivation. Oligodendrocyte lineage cells have a bidirectional response to MAPK signaling, where hyperactivating MAPK increases cell number and deletion reduces glial number. MAPK signaling activates several hundred downstream cues, but one of particular interest to this work is called Liver Kinase B1 (LKB1). LKB1 is a protein kinase which can regulate cell proliferation, survival, and metabolism. Here, I discovered that LKB1 is necessary for the development of parvalbumin expressing neurons. Collectively, these data identify disruption to certain ventral derivatives as a candidate pathogenic mechanism in neurodevelopmental conditions.

The GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the C9orf72 gene is the most common genetic abnormality associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastatingly progressive neurodegenerative diseases. The discovery of this genetic link confirmed that ALS and FTD reside along a spectrum with clinical and pathological commonalities. Historically understood as diseases resulting in neuronal death, the role of non-neuronal cells like astrocytes is still wholly unresolved. With evidence of cortical neurodegeneration leading to cognitive impairments in C9orf72-ALS/FTD, there is a need to investigate the role of cortical astrocytes in this disease spectrum. Here, a patient-derived induced pluripotent stem cell (iPSC) cortical astrocyte model was developed to investigate consequences of C9orf72-HRE pathogenic features in this cell type. Although there were no significant C9orf72-HRE pathogenic features in cortical astrocytes, transcriptomic, proteomic and phosphoproteomic profiles elucidated global disease-related phenotypes. Specifically, aberrant expression of astrocytic-synapse proteins and secreted factors were identified. SPARCL1, a pro-synaptogenic secreted astrocyte factor was found to be selectively decreased in C9orf72-ALS/FTD iPSC-cortical astrocytes. This finding was further validated in human tissue analyses, indicating that cortical astrocytes in C9orf72-ALS/FTD exhibit a reactive transformation that is characterized by a decrease in SPARCL1 expression. Considering the evidence for substantial astrogliosis and synaptic failure leading to cognitive impairments in C9orf72-ALS/FTD, these findings represent a novel understanding of how cortical astrocytes may contribute to the cortical neurodegeneration in this disease spectrum.

The RNA editing enzyme adenosine deaminase acting on double stranded RNA 2 (ADAR2) converts adenosine into inosine in regions of double stranded RNA. Here, it was discovered that this critical function of ADAR2 was dysfunctional in amyotrophic lateral sclerosis (ALS) mediated by the C9orf72 hexanucleotide repeat expansion, the most common genetic abnormality associated with ALS. Typically a nuclear protein, ADAR2 was localized in cytoplasmic accumulations in postmortem tissue from C9orf72 ALS patients. The mislocalization of ADAR2 was confirmed using immunostaining in a C9orf72 mouse model and motor neurons differentiated from C9orf72 patient induced pluripotent stem cells. Notably, the cytoplasmic accumulation of ADAR2 coexisted in neurons with cytoplasmic accumulations of TAR DNA binding protein 43 (TDP-43). Interestingly, ADAR2 overexpression in mammalian cell lines induced nuclear depletion and cytoplasmic accumulation of TDP-43, reflective of the pathology observed in ALS patients. The mislocalization of TDP-43 was dependent on the catalytic activity of ADAR2 and the ability of TDP-43 to bind directly to inosine containing RNA. In addition, TDP-43 nuclear export was significantly elevated in cells with increased RNA editing. Together these results describe a novel cellular mechanism by which alterations in RNA editing drive the nuclear export of TDP-43 leading to its cytoplasmic mislocalization. Considering the contribution of cytoplasmic TDP-43 to the pathogenesis of ALS, these findings represent a novel understanding of how the formation of pathogenic cytoplasmic TDP-43 accumulations may be initiated. Further research exploring this mechanism will provide insights into opportunities for novel therapeutic interventions.

Modeling sporadic amyotrophic lateral sclerosis (sALS) has been a challenge since there is no known single gene mutation that triggers disease pathogenesis. Although human induced pluripotent stem cells (hiPSCs) have created new opportunities in studying sALS, they do not retain important age associated phenotypic markers due to the rejuvenation stage that takes place during the reprogramming of somatic cells into hiPSCs. To overcome this obstacle, we performed an alternative method of direct neuronal conversion from patient fibroblasts that utilizes two transcription factors, Ngn2 and Ascl1. These transcription factors were sufficient to initiate direct neuronal conversion and produce induced neurons (iNeurons). Through the positive staining of neuronal markers Map2, Synapsin-1, and Human Nuclear Marker we found that induced neurons do display neuronal features that are seen in mature neurons.