Reversibility and developmental neuropathology of linear nevus sebaceous syndrome caused by dysregulation of the RAS pathway.

Linear nevus sebaceous syndrome (LNSS) is a neurocutaneous disorder caused by somatic gain-of-function mutations in KRAS or HRAS. LNSS brains have neurodevelopmental defects, including cerebral defects and epilepsy; however, its pathological mechanism and potentials for treatment are largely unclear. We show that introduction of KRASG12V in the developing mouse cortex results in subcortical nodular heterotopia and enhanced excitability, recapitulating major pathological manifestations of LNSS. Moreover, we show that decreased firing frequency of inhibitory neurons without KRASG12V expression leads to disrupted excitation and inhibition balance. Transcriptional profiling after destabilization domain-mediated clearance of KRASG12V in human neural progenitors and differentiating neurons identifies reversible functional networks underlying LNSS. Neurons expressing KRASG12V show molecular changes associated with delayed neuronal maturation, most of which are restored by KRASG12V clearance. These findings provide insights into the molecular networks underlying the reversibility of some of the neuropathologies observed in LNSS caused by dysregulation of the RAS pathway.

Labeling Dual Presynaptic Inputs using cFork Anterograde Tracing System.

Understanding brain function-related neural circuit connectivity is essential for investigating how cognitive functions are decoded in neural circuits. Trans-synaptic viral vectors are useful for identifying neural synaptic connectivity because of their ability to be transferred from transduced cells to synaptically connected cells. However, concurrent labeling of multisynaptic inputs to postsynaptic neurons is impossible with currently available trans-synaptic viral vectors. Here, we report a neural circuit tracing system that can simultaneously label postsynaptic neurons with two different markers, the expression of which is defined by presynaptic input connectivity. This system, called "cFork (see fork)", includes delivering serotype 1-packaged AAV vectors (AAV1s) containing Cre or flippase recombinase (FlpO) into two different presynaptic brain areas, and AAV5 with a dual gene expression cassette in postsynaptic neurons. Our in vitro and in vivo tests showed that selective expression of two different fluorescence proteins, EGFP and mScarlet, in postsynaptic neurons could be achieved by AAV1-mediated anterograde trans-synaptic transfer of Cre or FlpO constructs. When this tracing system was applied to the somatosensory barrel field cortex (S1BF) or striatum innervated by multiple presynaptic inputs, postsynaptic neurons defined by presynaptic inputs were simultaneously labeled with EGFP or mScarlet. Our new anterograde tracing tool may be useful for elucidating the complex multisynaptic connectivity of postsynaptic neurons regulating diverse brain functions.

Synaptic transmission and excitability during hypoxia with inflammation and reoxygenation in hippocampal CA1 neurons.

Although a number of experimental and clinical studies have shown that hypoxia typically accompanies acute inflammatory responses, the combinatorial effect of the two insults on basic neural function has not been thoroughly investigated. Previous studies have predominantly suggested that hypoxia reduces network activity; however, several studies suggest the opposite effect. Of note, inflammation is known to increase neural activity. In the current study, we examined the effects of limited oxygen in combination with an inflammatory stimulus, as well as the effects of reoxygenation, on synaptic transmission and excitability. We observed a significant reduction of both synaptic transmission and excitability when hypoxia and inflammation occurred in combination, whereas reoxygenation caused hyperexcitability of neurons. Further, we found that the observed reduction in synaptic transmission was due to compromised presynaptic release efficiency based on an adenosine-receptor-dependent increase in synaptic facilitation. Excitability changes in both directions were attributable to dynamic regulation of the hyperpolarization-activated cation current (Ih) and to changes in the input resistance and the voltage difference between resting membrane potential and action potential threshold. We found that zatebradine, an Ih current inhibitor, reduced the fluctuation in excitability, suggesting that it may have potential as a drug to ameliorate reperfusion brain injury.

An essential role for PICK1 in NMDA receptor-dependent bidirectional synaptic plasticity.

PICK1 is a calcium-sensing, PDZ domain-containing protein that interacts with GluR2 and GluR3 AMPA receptor (AMPAR) subunits and regulates their trafficking. Although PICK1 has been principally implicated in long-term depression (LTD), PICK1 overexpression in CA1 pyramidal neurons causes a CaMK- and PKC-dependent potentiation of AMPAR-mediated transmission and an increase in synaptic GluR2-lacking AMPARs, mechanisms associated with NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here, we directly tested whether PICK1 participates in both hippocampal NMDAR-dependent LTP and LTD. We show that the PICK1 potentiation of AMPAR-mediated transmission is NMDAR dependent and fully occludes LTP. Conversely, blockade of PICK1 PDZ interactions or lack of PICK1 prevents LTP. These observations demonstrate an important role for PICK1 in LTP. In addition, deletion of PICK1 or blockade of PICK1 PDZ binding prevented NMDAR-dependent LTD. Thus, PICK1 plays a critical role in bidirectional NMDAR-dependent long-term synaptic plasticity in the hippocampus.

Phosphatidylinositol phosphates as co-activators of Ca2+ binding to C2 domains of synaptotagmin 1.

Ca2+-dependent phospholipid binding to the C2A and C2B domains of synaptotagmin 1 is thought to trigger fast neurotransmitter release, but only Ca2+ binding to the C2B domain is essential for release. To investigate the underlying mechanism, we have compared the role of basic residues in Ca2+/phospholipid binding and in release. Mutations in a polybasic sequence on the side of the C2B domain beta-sandwich or in a basic residue in a top Ca2+-binding loop of the C2A domain (R233) cause comparable decreases in the apparent Ca2+ affinity of synaptotagmin 1 and the Ca2+ sensitivity of release, whereas mutation of the residue homologous to Arg233 in the C2B domain (Lys366) has no effect. Phosphatidylinositol polyphosphates co-activate Ca2+-dependent and -independent phospholipid binding to synaptotagmin 1, but the effects of these mutations on release only correlate with their effects on the Ca2+-dependent component. These results reveal clear distinctions in the Ca2+-dependent phospholipid binding modes of the synaptotagmin 1 C2 domains that may underlie their functional asymmetry and suggest that phosphatidylinositol polyphosphates may serve as physiological modulators of Ca2+ affinity of synaptotagmin 1 in vivo.

Alpha-synuclein regulates neuronal survival via Bcl-2 family expression and PI3/Akt kinase pathway.

Alpha-synuclein (alpha-SN) is a ubiquitous protein that is especially abundant in the brain and has been postulated to play a central role in the pathogenesis of Parkinson's disease, Alzheimer's disease, and other neurodegenerative disorders. However, little is known about the neuronal functions of alpha-SN and the molecular and cellular mechanisms underlying neuronal loss. Here, we show that alpha-SN plays dual roles of neuroprotection and neurotoxicity depending on its concentration or level of expression. At nanomolar concentrations, a-SN protected neurons against serum deprivation, oxidative stress, and excitotoxicity through the PI3/Akt signaling pathway, and its protective effect was increased by Bcl-2 overexpression. Conversely, at both low micromolar and overexpressed levels in the cell, alpha-SN resulted in cytotoxicity. This might be related to decreased Bcl-xL expression and increased bax expression, which is subsequently followed by cytochrome c release and caspase activation and also by microglia-mediated inflammatory responses via the NFkappaB and mitogen-activated protein kinase pathways.