The ERK1/2 cell signaling pathway is highly conserved and a prominent regulator of processes like cell proliferation, differentiation, and survival. During nervous system development, the ERK1/2 cascade is activated by the binding of growth factors to receptor tyrosine kinases, leading to the sequential phosphorylation of intracellular protein kinases in the pathway and eventually ERK1 and ERK2, the effectors of the pathway. Well-defined germline mutations resulting in hyperactive ERK1/2 signaling have been implicated in a group of neurodevelopmental disorders called RASopathies. RASopathic individuals often display features such as developmental delay, intellectual disability, cardio-facial abnormalities, and motor deficits. In addition, loss-of-function in ERK1/2 can lead to neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability. To better understand the pathology of these neurodevelopmental disorders, the role of ERK1/2 must be examined during the development of specific neuronal and glial subtypes. In this study, we bred transgenic mice with conditional deletion of ERK1/2 in cholinergic neuronal populations to investigate whether ERK1/2 mediates the survival or activity of basal forebrain and striatal cholinergic neurons during postnatal development. By postnatal day 10, we found that ERK1/2 did not seem to mediate cholinergic neuron number within the basal forebrain or striatum. In addition, we showed that expression of FosB, a neuronal activity-dependent transcription factor and target of ERK1/2, was not yet observed in cholinergic neurons within either of these anatomical regions by P10. Finally, our preliminary data suggested that FosB expression within layer IV of the somatosensory cortex, a target domain for basal forebrain cholinergic projections, also did not appear to be mediated by ERK1/2 signaling. However, since cholinergic neuron development is not yet complete by P10, future work should explore whether ERK1/2 plays any role in the long-term survival and function of basal forebrain and striatal cholinergic neurons in adulthood. This will hopefully provide more insight into the pathology of neurodevelopmental disorders and inform future therapeutic strategies.

Receptor-interacting serine/threonine protein kinase 1 (RIPK1) is an enzyme whose interaction with tumor necrosis factor receptor 1 (TNFR1) has been found to regulate cell death pathways, such as apoptosis and necroptosis, and neuroinflammation. Accumulating evidence in the past two decades has pointed to increased RIPK1 activity in various degenerative disorders, including Amyotrophic Lateral Sclerosis (ALS), stroke, traumatic brain injury (TBI) and Alzheimer’s Disease (AD). Given the work showing elevated RIPK1 in neurodegenerative disorders, to further understand the role of RIPK1 in disease pathogenesis, we created a conditional mouse overexpressing neuronal RIPK1 on a C57BL/6 background. These conditional transgenic mice overexpress murine RIPK1 under the CAMK2a neuronal promoter and the transgene is under the control of doxycycline. The removal of doxycycline turns on the RIPK1 transgene. Two cohorts of transgenic mice overexpressing neuronal RIPK1 (RIPK1 OE) were produced, and both had doxycycline removed at post-natal day 21. One cohort was behaviorally tested at 3-months-of-age and the second cohort was tested at 9-months-of-age. Behavioral testing included use of the RotaRod and the Morris water maze to assess motor coordination and spatial cognition, respectively. We found that the RIPK1 OE mice showed no deficits in motor coordination at either age but displayed spatial reference learning and memory deficits at 3- and 9-months-of-age. A subset of mice from two independent cohorts were utilized to assess RIPK1 levels and neuronal number. In these two cohorts of mice used for postmortem analysis, we found that at 3 months of age, ~2 months after transgene activation, RIPK1 levels are not higher in the hippocampus or cortex of the RIPK1 OE mice, however at 9 months, ~8 months after transgene activation, RIPK1 levels are significantly higher in the hippocampus and cortex of RIPK1 OE mice compared to the NonTg counterparts. A subset of tissue was stained against the neuronal marker NeuN. Using unbiased stereology to quantify hippocampal CA1 pyramidal neurons, we found no neuronal loss in the 3-month-old RIPK1 OE mice, but a 34.01% reduction in NeuN+ neuron count in 9-month-old RIPK1 OE mice. Collectively our data shows that RIPK1 overexpression impairs spatial reference learning and memory and reduces neuron number in the CA1 of the hippocampus, underlining the potential of RIPK1 as a target for ameliorating CNS pathology.