Chiung-Ya Chen

Tbr1 haploinsufficiency impairs amygdalar axonal projections and results in cognitive abnormality

The neuron-specific transcription factor T-box brain 1 (TBR1) regulates brain development. Disruptive mutations in the TBR1 gene have been repeatedly identified in patients with autism spectrum disorders (ASDs). Here, we show that Tbr1 haploinsufficiency results in defective axonal projections of amygdalar neurons and the impairment of social interaction, ultrasonic vocalization, associative memory and cognitive flexibility in mice. Loss of a copy of the Tbr1 gene altered the expression of Ntng1, Cntn2 and Cdh8 and reduced both inter- and intra-amygdalar connections. These developmental defects likely impair neuronal activation upon behavioral stimulation, which is indicated by fewer c-FOS–positive neurons and lack of GRIN2B induction in Tbr1+/− amygdalae. We also show that upregulation of amygdalar neuronal activity by local infusion of a partial NMDA receptor agonist, d-cycloserine, ameliorates the behavioral defects of Tbr1+/− mice. Our study suggests that TBR1 is important in the regulation of amygdalar axonal connections and cognition.

Sarm1, a negative regulator of innate immunity, interacts with syndecan-2 and regulates neuronal morphology

The syndecan Sds2 and the innate immunity inhibitor Sarm1 function together and in distinct pathways to promote proper neuronal morphogenesis.

CTTNBP2, but not CTTNBP2NL, regulates dendritic spinogenesis and synaptic distribution of the striatin–PP2A complex

Although both CTTNBP2 and CTTNBP2NL interact with cortactin and striatin/zinedin, CTTNBP2, but not CTTNBP2NL, is predominantly expressed in neurons and regulates dendritic spine distribution of cortactin and striatin/zinedin. The finding may be relevant to the association of CTTNBP2 with autism.

TLR7 Negatively Regulates Dendrite Outgrowth through the Myd88–c-Fos–IL-6 Pathway

Toll-like receptors (TLRs) recognize both pathogen- and danger-associated molecular patterns and induce innate immune responses. Some TLRs are expressed in neurons and regulate neurodevelopment and neurodegeneration. However, the downstream signaling pathways and effectors for TLRs in neurons are still controversial. In this report, we provide evidence that TLR7 negatively regulates dendrite growth through the canonical myeloid differentiation primary response gene 88 (Myd88)–c-Fos–interleukin (IL)-6 pathway. Although both TLR7 and TLR8 recognize single-stranded RNA (ssRNA), the results of quantitative reverse transcription-PCR suggested that TLR7 is the major TLR recognizing ssRNA in brains. In both in vitro cultures and in utero electroporation experiments, manipulation of TLR7 expression levels was sufficient to alter neuronal morphology, indicating the presence of intrinsic TLR7 ligands. Besides, the RNase A treatment that removed ssRNA in cultures promoted dendrite growth. We also found that the addition of ssRNA and synthetic TLR7 agonists CL075 and loxoribine, but not R837 (imiquimod), to cultured neurons specifically restricted dendrite growth via TLR7. These results all suggest that TLR7 negatively regulates neuronal differentiation. In cultured neurons, TLR7 activation induced IL-6 and TNF-α expression through Myd88. Using Myd88-, IL-6-, and TNF-α-deficient neurons, we then demonstrated the essential roles of Myd88 and IL-6, but not TNF-α, in the TLR7 pathway to restrict dendrite growth. In addition to neuronal morphology, TLR7 knockout also affects mouse behaviors, because young mutant mice ∼2 weeks of age exhibited noticeably lower exploratory activity in an open field. In conclusion, our study suggests that TLR7 negatively regulates dendrite growth and influences cognition in mice.

Bcl11A/CTIP1 mediates the effect of the glutamate receptor on axon branching and dendrite outgrowth.

J. Neurochem. (2010) 114, 1381–1392.

Neuronally-expressed Sarm1 regulates expression of inflammatory and antiviral cytokines in brains:

Sarm1 is the fifth Toll/IL-1 receptor (TIR) domain-containing adaptor protein identified to regulate TLR downstream signaling. Unlike the other TIR domain-containing adaptor proteins, Sarm1 is predominantly expressed in the brain. Our previous study indicated that Sarm1 regulates dendritic growth, axonal extension and neuronal polarity. Here, we investigated whether Sarm1 is involved in innate immunity in the brain. First, regional and cell-type distribution of Sarm1 in mouse brains was revealed using double immunostaining. Sarm1 was widely distributed in different regions of brains, including the cerebral cortex, hippocampus, amygdala, cerebellum and midbrain. Moreover, Sarm1 is present in both projection and inhibitory neurons, but, interestingly, not in microglial cells—the main immune cells in the brain. These results suggest that Sarm1 is unlikely to regulate microglial activity in a cell-autonomous manner. However, compared with wild type littermates, the RNA expression levels of several inflammatory and antiviral cytokines were altered in the embryonic and adult brains of Sarm1 knockdown transgenic mice. These data imply that Sarm1 influences cytokine expression in neurons. In conclusion, our findings suggest that Sarm1 regulates the innate immune responses of the central nervous system through regulating the inflammatory and anti-virus cytokines produced by neurons.

Innate immune responses regulate morphogenesis and degeneration: roles of Toll-like receptors and Sarm1 in neurons

The central nervous system is recognized as an immunoprivileged site because peripheral immune cells do not typically enter it. Microglial cells are thought to be the main immune cells in brain. However, recent reports have indicated that neurons express the key players of innate immunity, including Toll-like receptors (TLRs) and their adaptor proteins (Sarm1, Myd88, and Trif), and may produce cytokines in response to pathogen infection. In the absence of an immune challenge, neuronal TLRs can detect intrinsic danger signals and modulate neuronal morphology and function. In this article, we review the recent findings on the involvement of TLRs and Sarm1 in controlling neuronal morphogenesis and neurodegeneration. Abnormal behaviors in TLR- and Sarm1-deficient mice are also discussed.

Endothelin‐Mediated Calcium Response and Inositol 1,4,5‐Trisphosphate Release in Neuroblastoma‐Glioma Hybrid Cells (NG108‐15): Cross Talk with ATP and Bradykinin

Abstract: Addition of endothelins (ETs) to neuroblastomaglioma hybrid cells (NG108‐15) induced increases in cytosolic free Ca2+ ([Ca2+]i) levels of labeled inositol monophosphates and inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3]. The increases in [Ca2+]i elicited by the three ETs (ET‐1, ET‐2, and ET‐3) were transient and did not show a sustained phase. Chelating extracellular Ca2+ in the medium by adding excess EGTA decreased the ET‐mediated Ca2+ response by 40‐50%. This result indicates that a substantial portion of the increase in [Ca2+]i was due to influx from an extracellular source. However, the increase in [Ca2+]i was not affected by verapamil or nifedipine (10−5M). A rank order potency of ET‐1 ET‐2 ET‐3 is shown for the stimulated increase in [Ca2+]i, as well as labeled inositol phosphates, in these cells. ATP (10−4M) and bradykinin (10−7M) also induced the increases in [Ca2+]i and Ins(1,4,5)P3 in NG108‐15 cells, albeit to a different extent. When compared at 10−7M, bradykinin elicited a five‐ to sixfold higher increase in the level of Ins(1,4,5)P3, but less than a twofold higher increase in [Ca2+]i than those induced by ET‐1. Additive increases in both Ins(1,4,5)P3 and [Ca2+]i were observed when ET‐1, ATP, and bradykinin were added to the cells in different combinations, suggesting that each receptor agonist is responsible for the hydrolysis of a pool of polyphosphoinositide within the membrane. ET‐1 exhibited homologous desensitization of the Ca2+ response, but partial heterologous desensitization to the Ca2+ response elicited by ATP. On the contrary, ET‐1 did not desensitize the response elicited by bradykinin, although bradykinin exhibited complete heterologous desensitization to the response elicited by ET‐1. Taken together, these results illustrate that, in NG108‐15 cells, a considerable amount of receptor cross talk occurs between ET and other receptors that transmit signals through the polyphosphoinositide pathway.

Sarm1 deficiency impairs synaptic function and leads to behavioral deficits, which can be ameliorated by an mGluR allosteric modulator

Innate immune responses have been shown to influence brain development and function. Dysregulation of innate immunity is significantly associated with psychiatric disorders such as autism spectrum disorders and schizophrenia, which are well-known neurodevelopmental disorders. Recent studies have revealed that critical players of the innate immune response are expressed in neuronal tissues and regulate neuronal function and activity. For example, Sarm1, a negative regulator that acts downstream of Toll-like receptor (TLR) 3 and 4, is predominantly expressed in neurons. We have previously shown that Sarm1 regulates neuronal morphogenesis and the expression of inflammatory cytokines in the brain, which then affects learning ability, cognitive flexibility, and social interaction. Because impaired neuronal morphogenesis and dysregulation of cytokine expression may disrupt neuronal activity, we investigated whether Sarm1 knockdown affects the synaptic responses of neurons. We here show that reduced Sarm1 expression impairs metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) formation but enhances N-methyl-D-aspartate receptor (NMDAR)-dependent long-term potentiation production in hippocampal CA1 neurons. The expression levels of post-synaptic proteins, including NR2a, NR1, Shank1 and Shank3, are also altered in Sarm1 knockdown mice, suggesting a role for Sarm1 in the maintenance of synaptic homeostasis. The addition of a positive allosteric modulator of mGluR5, CDPPB, ameliorates the LTD defects in slice recording and the behavioral deficits in social interaction and associative memory. These results suggest an important role for mGluR5 signaling in the function of Sarm1. In conclusion, our study demonstrates a role for Sarm1 in the regulation of synaptic plasticity. Through these mechanisms, Sarm1 knockdown results in the impairment of associative memory and social interactions in mice.

TLR3 downregulates expression of schizophrenia gene Disc1 via MYD88 to control neuronal morphology.

Viral infection during fetal or neonatal stages increases the risk of developing neuropsychiatric disorders such as schizophrenia and autism spectrum disorders. Although neurons express several key regulators of innate immunity, the role of neuronal innate immunity in psychiatric disorders is still unclear. Using cultured neurons and in vivo mouse brain studies, we show here that Toll‐like receptor 3 (TLR3) acts through myeloid differentiation primary response gene 88 (MYD88) to negatively control Disrupted in schizophrenia 1 (Disc1) expression, resulting in impairment of neuronal development. Cytokines are not involved in TLR3‐mediated inhibition of dendrite outgrowth. Instead, TLR3 signaling suppresses expression of several psychiatric disorder‐related genes, including Disc1. The impaired dendritic arborization caused by TLR3 activation is rescued by MYD88 deficiency or DISC1 overexpression. In addition, TLR3 activation at the neonatal stage increases dendritic spine density, but narrows spine heads at postnatal day 21 (P21), suggesting a long‐lasting effect of TLR3 activation on spinogenesis. Our study reveals a novel mechanism of TLR3 in regulation of dendritic morphology and provides an explanation for how environmental factors influence mental health.