Cortical Neuron Migration and Dendrite Morphology are Regulated by Carboxypeptidase E.

Higher brain function relies on proper development of the cerebral cortex, including correct positioning of neurons and dendrite morphology. Disruptions in these processes may result in various neurocognitive disorders. Mutations in the CPE gene, which encodes carboxypeptidase E (CPE), have been linked to depression and intellectual disability. However, it remains unclear whether CPE is involved in early brain development and in turn contributes to the pathophysiology of neurocognitive disorders. Here, we investigate the effects of CPE knockdown on early brain development and explore the functional significance of the interaction between CPE and its binding partner p150Glued. We demonstrate that CPE is required for cortical neuron migration and dendrite arborization. Furthermore, we show that expression of CPE-C10 redistributes p150Glued from the centrosome and that disruption of CPE interaction with p150Glued leads to abnormal neuronal migration and dendrite morphology, suggesting that a complex between CPE and p150Glued is necessary for proper neurodevelopment.

Lipids and phosphates at odds in synaptic depression.

Long-term depression (LTD) is a reduction in the efficacy of neuronal synapses, but the molecular basis of LTD signaling and how these signals lead to phenotypic outcomes, such as the shrinkage of synaptic regions, is not clear. In a new report, Woolfrey et al use chemically-induced LTD and a multitude of in vitro biochemical assays to provide evidence that synaptic removal of the scaffolding protein AKAP79/150 promotes LTD-induced spine shrinkage. The further identification of CaMKII, a kinase primarily associated with long-term potentiation (LTP), as a requirement for AKAP79/150 removal, uncovers unexpected interplay between different post-translational modifications and points to a new model of LTD.

d-Serine administration affects nitric oxide synthase 1 adaptor protein and DISC1 expression in sex-specific manner.

Antipsychotic medications are inefficient at treating symptoms of schizophrenia (SCZ), and N-methyl d-aspartate receptor (NMDAR) agonists are potential therapeutic alternatives. As such, these agonists may act on different pathways and proteins altered in the brains of patients with SCZ than do antipsychotic medications. Here, we investigate the effects of administration of the antipsychotic haloperidol and NMDAR agonist d-serine on function and expression of three proteins that play significant roles in SCZ: nitric oxide synthase 1 adaptor protein (NOS1AP), dopamine D2 (D2) receptor, and disrupted in schizophrenia 1 (DISC1). We administered haloperidol or d-serine to male and female Sprague Dawley rats via intraperitoneal injection for 12 days and subsequently examined cortical expression of NOS1AP, D2 receptor, and DISC1. We found sex-specific effects of haloperidol and d-serine treatment on the expression of these proteins. Haloperidol significantly reduced expression of D2 receptor in male, but not female, rats. Conversely, d-serine reduced expression of NOS1AP in male rats and did not affect D2 receptor expression. d-serine treatment also reduced expression of DISC1 in male rats and increased DISC1 expression in female rats. As NOS1AP is overexpressed in the cortex of patients with SCZ and negatively regulates NMDAR signaling, we subsequently examined whether treatment with antipsychotics or NMDAR agonists can reverse the detrimental effects of NOS1AP overexpression in vitro as previously reported by our group. NOS1AP overexpression promotes reduced dendrite branching in vitro, and as such, we treated cortical neurons overexpressing NOS1AP with different antipsychotics (haloperidol, clozapine, fluphenazine) or d-serine for 24 h and determined the effects of these drugs on NOS1AP expression and dendrite branching. While antipsychotics did not affect NOS1AP protein expression or dendrite branching in vitro, d-serine reduced NOS1AP expression and rescued NOS1AP-mediated reductions in dendrite branching. Taken together, our data suggest that d-serine influences the function and expression of NOS1AP, D2 receptor, and DISC1 in a sex-specific manner and reverses the effects of NOS1AP overexpression on dendrite morphology.

Microfluidic platforms for the study of neuronal injury in vitro.

Traumatic brain injury (TBI) affects 5.3 million people in the United States, and there are 12,500 new cases of spinal cord injury (SCI) every year. There is yet a significant need for in vitro models of TBI and SCI in order to understand the biological mechanisms underlying central nervous system (CNS) injury and to identify and test therapeutics to aid in recovery from neuronal injuries. While TBI or SCI studies have been aided with traditional in vivo and in vitro models, the innate limitations in specificity of injury, isolation of neuronal regions, and reproducibility of these models can decrease their usefulness in examining the neurobiology of injury. Microfluidic devices provide several advantages over traditional methods by allowing researchers to (1) examine the effect of injury on specific neural components, (2) fluidically isolate neuronal regions to examine specific effects on subcellular components, and (3) reproducibly create a variety of injuries to model TBI and SCI. These microfluidic devices are adaptable for modeling a wide range of injuries, and in this review, we will examine different methodologies and models recently utilized to examine neuronal injury. Specifically, we will examine vacuum-assisted axotomy, physical injury, chemical injury, and laser-based axotomy. Finally, we will discuss the benefits and downsides to each type of injury model and discuss how researchers can use these parameters to pick a particular microfluidic device to model CNS injury.

Time-dependent homeostatic mechanisms underlie brain-derived neurotrophic factor action on neural circuitry.

Plasticity and homeostatic mechanisms allow neural networks to maintain proper function while responding to physiological challenges. Despite previous work investigating morphological and synaptic effects of brain-derived neurotrophic factor (BDNF), the most prevalent growth factor in the central nervous system, how exposure to BDNF manifests at the network level remains unknown. Here we report that BDNF treatment affects rodent hippocampal network dynamics during development and recovery from glutamate-induced excitotoxicity in culture. Importantly, these effects are not obvious when traditional activity metrics are used, so we delve more deeply into network organization, functional analyses, and in silico simulations. We demonstrate that BDNF partially restores homeostasis by promoting recovery of weak and medium connections after injury. Imaging and computational analyses suggest these effects are caused by changes to inhibitory neurons and connections. From our in silico simulations, we find that BDNF remodels the network by indirectly strengthening weak excitatory synapses after injury. Ultimately, our findings may explain the difficulties encountered in preclinical and clinical trials with BDNF and also offer information for future trials to consider.

Glutamate Receptors Mediate Changes to Dendritic Mitochondria in Neurons Grown on Stiff Substrates.

The stiffness of brain tissue changes during development and disease. These changes can affect neuronal morphology, specifically dendritic arborization. We previously reported that N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors regulate dendrite number and branching in a manner that is dependent on substrate stiffness. Since mitochondria affect the shape of dendrites, in this study, we determined whether the stiffness of substrates on which rat hippocampal neurons are grown affects mitochondrial characteristics and if glutamate receptors mediate the effects of substrate stiffness. Dendritic mitochondria are small, short, simple, and scarce in neurons cultured on substrates of 0.5 kPa stiffness. In contrast, dendritic mitochondria are large, long, complex, and low in number in neurons grown on substrates of 4 kPa stiffness. Dendritic mitochondria of neurons cultured on glass are high in number and small with complex shapes. Treatment of neurons grown on the stiffer gels or glass with the NMDA and AMPA receptor antagonists, 2-amino-5-phosphonopentanoic acid and 6-cyano-7-nitroquinoxaline-2,3-dione, respectively, results in mitochondrial characteristics of neurons grown on the softer substrate. These results suggest that glutamate receptors play important roles in regulating both mitochondrial morphology and dendritic arborization in response to substrate stiffness.

Current advances in in vitro models of central nervous system trauma.

CNS trauma is a prominent cause of mortality and morbidity, and although much effort has focused on developing treatments for CNS trauma-related pathologies, little progress has been made. Pre-clinical models of TBI and SCI suffer from significant drawbacks, which result in substantial failures during clinical translation of promising pre-clinical therapies. Here, we review recent advances made in the development of in vitro models of CNS trauma, the promises and drawbacks of current in vitro CNS injury models, and the attributes necessary for future models to accurately mimic the trauma microenvironment and facilitate CNS trauma drug discovery. The goal is to provide insight for the development of future CNS injury models and to aid researchers in selecting effective models for pre-clinical research of trauma therapeutics.

Interaction Between CRIPT and PSD-95 Is Required for Proper Dendritic Arborization in Hippocampal Neurons.

CRIPT, the cysteine-rich PDZ-binding protein, binds to the third PDZ domain of PSD-95 (postsynaptic density protein 95) family proteins and directly binds microtubules, linking PSD-95 family proteins to the neuronal cytoskeleton. Here, we show that overexpression of a full-length CRIPT leads to a modest decrease, and knockdown of CRIPT leads to an increase in dendritic branching in cultured rat hippocampal neurons. Overexpression of truncated CRIPT lacking the PDZ domain-binding motif, which does not bind to PSD-95, significantly decreases dendritic arborization. Conversely, overexpression of a full-length CRIPT significantly increases the number of immature and mature dendritic spines, and this effect is not observed when CRIPT∆PDZ is overexpressed. Competitive inhibition of CRIPT binding to the third PDZ domain of PSD-95 with PDZ3-binding peptides resulted in differential effects on dendritic arborization based on the origin of respective peptide sequence. These results highlight multifunctional roles of CRIPT during development and underscore the significance of the interaction between CRIPT and the third PDZ domain of PSD-95.

Axonal Development: RhoA Restrains but Does Not Specify.

Neurons develop polarity by the formation of specialized dendritic and axonal structural compartments. A new report now provides evidence that reveals how neurons regulate the initiation and further maintenance of axonal growth, challenging our currently held view of RhoA function in axogenesis.

Caffeine inhibits hypoxia-induced nuclear accumulation in HIF-1α and promotes neonatal neuronal survival.

Apnea of prematurity (AOP) defined as cessation of breathing for 15-20 s, is commonly seen in preterm infants. Caffeine is widely used to treat AOP due to its safety and effectiveness. Caffeine releases respiratory arrest by competing with adenosine for binding to adenosine A1 and A2A receptors (A1R and A2AR). Long before its use in treating AOP, caffeine has been used as a psychostimulant in adult brains. However, the effect of caffeine on developing brains remains unclear. We found that A1R proteins for caffeine binding were expressed in the brains of neonatal rodents and preterm infants (26-27 weeks). Neonatal A1R proteins colocalized with PSD-95, suggesting its synaptic localization. In contrast, our finding on A2R expression in neonatal neurons was restricted to the mRNA level as detected by single cell RT/PCR due to the lack of specific A2AR antibody. Furthermore, caffeine (200 µM) at a dose twice higher than the clinically relevant dose (36-130 µM) had minor or no effects on several basic neuronal functions, such as neurite outgrowth, synapse formation, expression of A1R and transcription of CREB-1 and c-Fos, further supporting the safety of caffeine for clinical use. We found that treatment with CoCl2 (125 µM), a hypoxia mimetic agent, for 24 h triggered neuronal death and nuclear accumulation of HIF-1α in primary neuronal cultures. Subsequent treatment with caffeine at a concentration of 100 µM alleviated CoCl2-induced cell death and prevented nuclear accumulation of HIF-1α. Consistently, caffeine treatment in early postnatal life of neonatal mice (P4-P7) also prevented subsequent hypoxia-induced nuclear increase of HIF-1α. Together, our data support the utility of caffeine in alleviating hypoxia-induced damages in developing neurons.

Dynamin and reverse-mode sodium calcium exchanger blockade confers neuroprotection from diffuse axonal injury.

Mild traumatic brain injury (mTBI) is a frequently overlooked public health concern that is difficult to diagnose and treat. Diffuse axonal injury (DAI) is a common mTBI neuropathology in which axonal shearing and stretching induces breakdown of the cytoskeleton, impaired axonal trafficking, axonal degeneration, and cognitive dysfunction. DAI is becoming recognized as a principal neuropathology of mTBI with supporting evidence from animal model, human pathology, and neuroimaging studies. As mitochondrial dysfunction and calcium overload are critical steps in secondary brain and axonal injury, we investigated changes in protein expression of potential targets following mTBI using an in vivo controlled cortical impact model. We show upregulated expression of sodium calcium exchanger1 (NCX1) in the hippocampus and cortex at distinct time points post-mTBI. Expression of dynamin-related protein1 (Drp1), a GTPase responsible for regulation of mitochondrial fission, also changes differently post-injury in the hippocampus and cortex. Using an in vitro model of DAI previously reported by our group, we tested whether pharmacological inhibition of NCX1 by SN-6 and of dynamin1, dynamin2, and Drp1 by dynasore mitigates secondary damage. Dynasore and SN-6 attenuate stretch injury-induced swelling of axonal varicosities and mitochondrial fragmentation. In addition, we show that dynasore, but not SN-6, protects against H2O2-induced damage in an organotypic oxidative stress model. As there is currently no standard treatment to mitigate cell damage induced by mTBI and DAI, this work highlights two potential therapeutic targets for treatment of DAI in multiple models of mTBI and DAI.

The 3' UTRs of Brain-Derived Neurotrophic Factor Transcripts Differentially Regulate the Dendritic Arbor.

The patterning of dendrites is regulated by many factors, such as brain-derived neurotrophic factor (BDNF), which our laboratory has previously shown alters the dendritic arbor uniquely depending on the mode of extracellular application. In the current work, we examine how BDNF affects dendritogenesis in hippocampal neurons when it is overexpressed intracellularly by transcripts previously reported to be transported to distinct cellular compartments. The BDNF gene is processed at two different polyadenylation sites, leading to mRNA transcription with two different length 3' untranslated regions (UTRs), and therefore, different mRNA localization preferences. We found that overexpression of BDNF mRNA with or without 3' UTRs significantly alters dendritic branching compared to branching in control neurons as analyzed by Sholl distribution curves. Unexpectedly, we found that the overexpression of the shorter BDNF mRNA (reported to be preferentially targeted to the cell body) results in similar changes to Sholl curves compared to overexpression of the longer BDNF mRNA (reported to be preferentially targeted to both the cell body and dendrites). We also investigated whether the BDNF receptor TrkB mediates these changes and found that inhibiting TrkB blocks increases in Sholl curves, although at different distances depending on the transcript's UTR. Finally, although it is not found in nature, we also examined the effects of overexpressing BDNF mRNA with the unique portion of the longer 3' UTR since it was previously shown to be necessary for dendritic targeting of mRNA. We found that its overexpression increases Sholl curves at distances close to the cell body and that these changes also depend on TrkB activity. This work illustrates how the mRNA spatial code affects how BDNF alters local dendritogenesis and how TrkB may mediate these effects. Finally, our findings emphasize the importance of intracellular transport of BDNF mRNAs in the regulation of dendrite morphology.