SHP2 regulates GluA2 tyrosine phosphorylation required for AMPA receptor endocytosis and mGluR-LTD.

Posttranslational modifications regulate the properties and abundance of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that mediate fast excitatory synaptic transmission and synaptic plasticity in the central nervous system. During long-term depression (LTD), protein tyrosine phosphatases (PTPs) dephosphorylate tyrosine residues in the C-terminal tail of AMPA receptor GluA2 subunit, which is essential for GluA2 endocytosis and group I metabotropic glutamate receptor (mGluR)-dependent LTD. However, as a selective downstream effector of mGluRs, the mGluR-dependent PTP responsible for GluA2 tyrosine dephosphorylation remains elusive at Schaffer collateral (SC)-CA1 synapses. In the present study, we find that mGluR5 stimulation activates Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) by increasing phospho-Y542 levels in SHP2. Under steady-state conditions, SHP2 plays a protective role in stabilizing phospho-Y869 of GluA2 by directly interacting with GluA2 phosphorylated at Y869, without affecting GluA2 phospho-Y876 levels. Upon mGluR5 stimulation, SHP2 dephosphorylates GluA2 at Y869 and Y876, resulting in GluA2 endocytosis and mGluR-LTD. Our results establish SHP2 as a downstream effector of mGluR5 and indicate a dual action of SHP2 in regulating GluA2 tyrosine phosphorylation and function. Given the implications of mGluR5 and SHP2 in synaptic pathophysiology, we propose SHP2 as a promising therapeutic target for neurodevelopmental and autism spectrum disorders.

Cerebellar nuclei neurons projecting to the lateral parabrachial nucleus modulate classical fear conditioning.

Multiple brain regions are engaged in classical fear conditioning. Despite evidence for cerebellar involvement in fear conditioning, the mechanisms by which cerebellar outputs modulate fear learning and memory remain unclear. We identify a population of deep cerebellar nucleus (DCN) neurons with monosynaptic glutamatergic projections to the lateral parabrachial nucleus (lPBN) (DCN→lPBN neurons) in mice. While optogenetic suppression of DCN→lPBN neurons impairs auditory fear memory, activation of DCN→lPBN neurons elicits freezing behavior only after auditory fear conditioning. Moreover, auditory fear conditioning potentiates DCN-lPBN synapses, and subsequently, auditory cue activates lPBN neurons after fear conditioning. Furthermore, DCN→lPBN neuron activation can replace the auditory cue but not footshock in fear conditioning. These findings demonstrate that cerebellar nuclei modulate auditory fear conditioning via transmitting conditioned stimuli signals to the lPBN. Collectively, our findings suggest that the DCN-lPBN circuit is a part of neuronal substrates within interconnected brain regions underscoring auditory fear memory.

Neuron type-specific expression of a mutant KRAS impairs hippocampal-dependent learning and memory.

KRAS mutations are associated with rare cases of neurodevelopmental disorders that can cause intellectual disabilities. Previous studies showed that mice expressing a mutant KRAS have impaired the development and function of GABAergic inhibitory neurons, which may contribute to behavioural deficits in the mutant mice. However, the underlying cellular mechanisms and the role of excitatory neurons in these behavioural deficits in adults are not fully understood. Herein, we report that neuron type-specific expression of a constitutively active mutant KRASG12V in either excitatory or inhibitory neurons resulted in spatial memory deficits in adult mice. In inhibitory neurons, KRASG12V induced ERK activation and enhanced GABAergic synaptic transmission. Expressing KRASG12V in inhibitory neurons also impaired long-term potentiation in the hippocampal Shaffer-collateral pathway, which could be rescued by picrotoxin treatment. In contrast, KRASG12V induced ERK activation and neuronal cell death in excitatory neurons, which might have contributed to the severe behavioural deficits. Our results showed that both excitatory and inhibitory neurons are involved in mutant KRAS-associated learning deficits in adults via distinct cellular mechanisms.

Persistent Activity of Metabotropic Glutamate Receptor 5 in the Periaqueductal Gray Constrains Emergence of Chronic Neuropathic Pain.

Pain sensation is powerfully modulated by signal processing in the brain, and pain becomes chronic with the dysfunction of the pain modulatory system; however, the underlying mechanisms are unclear. We found that the metabotropic glutamate receptor 5 (mGluR5) in the periaqueductal gray (PAG), the key area of endogenous pain modulation, is persistently active in normal conditions to maintain an appropriate sensory perception. In the neuropathic pain condition, Homer1a, an activity-dependent immediate early gene product, disrupted the persistent mGluR5 activity resulting in chronic pain. Remarkably a single-time blockage of the mGluR5 resulted in chronic neuropathic pain-like symptoms even in the absence of nerve injury. The decline of mGluR5 activity induced the pain modulatory dysfunction with a profound reduction of excitability of PAG neurons. These findings uncover the role of the persistent mGluR5 activity in vivo and provide new insight into how pain becomes chronic with the maladaptive coping of the PAG to pain sensation.

Connecting the dots between SHP2 and glutamate receptors.

SHP2 is an unusual protein phosphatase that functions as an activator for several signaling pathways, including the RAS pathway, while most other phosphatases suppress their downstream signaling cascades. The physiological and pathophysiological roles of SHP2 have been extensively studied in the field of cancer research. Mutations in the PTPN11 gene which encodes SHP2 are also highly associated with developmental disorders, such as Noonan syndrome (NS), and cognitive deficits including learning disabilities are common among NS patients. However, the molecular and cellular mechanism by which SHP2 is involved in cognitive functions is not well understood. Recent studies using SHP2 mutant mice or pharmacological inhibitors have shown that SHP2 plays critical role in learning and memory and synaptic plasticity. Here, we review the recent studies demonstrating that SHP2 is involved in synaptic plasticity, and learning and memory, by the regulation of the expression and/or function of glutamate receptors. We suggest that each cell type may have distinct paths connecting the dots between SHP2 and glutamate receptors, and these paths may also change with aging.

Enriched expression of NF1 in inhibitory neurons in both mouse and human brain.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by loss-of-function mutations in NF1 gene, which encodes a GTPase activating protein for RAS. NF1 affects multiple systems including brain and is highly associated with cognitive deficits such as learning difficulties and attention deficits. Previous studies have suggested that GABAergic inhibitory neuron is the cell type primarily responsible for the learning deficits in mouse models of NF1. However, it is not clear how NF1 mutations selectively affect inhibitory neurons in the central nervous system. In this study, we show that the expression level of Nf1 is significantly higher in inhibitory neurons than in excitatory neurons in mouse hippocampus and cortex by using in situ hybridization. Furthermore, we also found that NF1 is enriched in inhibitory neurons in the human cortex, confirming that the differential expressions of NF1 between two cell types are evolutionarily conserved. Our results suggest that the enriched expression of NF1 in inhibitory neurons may underlie inhibitory neuron-specific deficits in NF1.

Excitatory neuron-specific SHP2-ERK signaling network regulates synaptic plasticity and memory.

Mutations in RAS signaling pathway components cause diverse neurodevelopmental disorders, collectively called RASopathies. Previous studies have suggested that dysregulation in RAS-extracellular signal-regulated kinase (ERK) activation is restricted to distinct cell types in different RASopathies. Some cases of Noonan syndrome (NS) are associated with gain-of-function mutations in the phosphatase SHP2 (encoded by PTPN11); however, SHP2 is abundant in multiple cell types, so it is unclear which cell type(s) contribute to NS phenotypes. Here, we found that expressing the NS-associated mutant SHP2D61G in excitatory, but not inhibitory, hippocampal neurons increased ERK signaling and impaired both long-term potentiation (LTP) and spatial memory in mice, although endogenous SHP2 was expressed in both neuronal types. Transcriptomic analyses revealed that the genes encoding SHP2-interacting proteins that are critical for ERK activation, such as GAB1 and GRB2, were enriched in excitatory neurons. Accordingly, expressing a dominant-negative mutant of GAB1, which reduced its interaction with SHP2D61G, selectively in excitatory neurons, reversed SHP2D61G-mediated deficits. Moreover, ectopic expression of GAB1 and GRB2 together with SHP2D61G in inhibitory neurons resulted in ERK activation. These results demonstrate that RAS-ERK signaling networks are notably different between excitatory and inhibitory neurons, accounting for the cell type-specific pathophysiology of NS and perhaps other RASopathies.

Vascular and Neurogenic Rejuvenation in Aging Mice by Modulation of ASM.

Although many reports have revealed dysfunction of endothelial cells in aging, resulting in blood-brain barrier (BBB) breakdown, the underlying mechanism or mechanisms remain to be explored. Here, we find that acid sphingomyelinase (ASM) is a critical factor for regulating brain endothelial barrier integrity. ASM is increased in brain endothelium and/or plasma of aged humans and aged mice, leading to BBB disruption by increasing caveolae-mediated transcytosis. Genetic inhibition and endothelial-specific knockdown of ASM in mice ameliorated BBB breakdown and neurocognitive impairment during aging. Using primary mouse brain endothelial cells, we found that ASM regulated the caveolae-cytoskeleton interaction through protein phosphatase 1-mediated ezrin/radixin/moesin (ERM) dephosphorylation and apoptosis. Moreover, mice with conditional ASM overexpression in brain endothelium accelerated significant BBB impairment and neurodegenerative change. Overall, these results reveal a novel role for ASM in the control of neurovascular function in aging, suggesting that ASM may represent a new therapeutic target for anti-aging.

SPIN90 Modulates Long-Term Depression and Behavioral Flexibility in the Hippocampus.

The importance of actin-binding proteins (ABPs) in the regulation of synapse morphology and plasticity has been well established. SH3 protein interacting with Nck, 90 kDa (SPIN90), an Nck-interacting protein highly expressed in synapses, is essential for actin remodeling and dendritic spine morphology. Synaptic targeting of SPIN90 to spine heads or dendritic shafts depends on its phosphorylation state, leading to blockage of cofilin-mediated actin depolymerization and spine shrinkage. However, the physiological role of SPIN90 in long-term plasticity, learning and memory are largely unknown. In this study, we demonstrate that Spin90-knockout (KO) mice exhibit substantial deficits in synaptic plasticity and behavioral flexibility. We found that loss of SPIN90 disrupted dendritic spine density in CA1 neurons of the hippocampus and significantly impaired long-term depression (LTD), leaving basal synaptic transmission and long-term potentiation (LTP) intact. These impairments were due in part to deficits in AMPA receptor endocytosis and its pre-requisites, GluA1 dephosphorylation and postsynaptic density (PSD) 95 phosphorylation, but also by an intrinsic activation of Akt-GSK3ß signaling as a result of Spin90-KO. In accordance with these defects, mice lacking SPIN90 were found to carry significant deficits in object-recognition and behavioral flexibility, while learning ability was largely unaffected. Collectively, these findings demonstrate a novel modulatory role for SPIN90 in hippocampal LTD and behavioral flexibility.

STIM1 Regulates Somatic Ca2+ Signals and Intrinsic Firing Properties of Cerebellar Purkinje Neurons.

Control of Ca2+ flux between the cytosol and intracellular Ca2+ stores is essential for maintaining normal cellular function. It has been well established in both neuronal and non-neuronal cells that stromal interaction molecule 1 (STIM1) initiates and regulates refilling Ca2+ into the ER. Here, we describe a novel, additional role for STIM1, the regulation of free cytosolic Ca2+, and the consequent control of spike firing in neurons. Among central neurons, cerebellar Purkinje neurons express the highest level of STIM1, and they fire continuously in the absence of stimulation, making somatic Ca2+ homeostasis of particular importance. By using Purkinje neuron-specific STIM1 knock-out (STIM1PKO) male mice, we found that the deletion of STIM1 delayed clearance of cytosolic Ca2+ in the soma during ongoing neuronal firing. Deletion of STIM1 also reduced the Purkinje neuronal excitability and impaired intrinsic plasticity without affecting long-term synaptic plasticity. In vestibulo-ocular reflex learning, STIM1PKO male mice showed severe deficits in memory consolidation, whereas they were normal in memory acquisition. Our results suggest that STIM1 is critically involved in the regulation of the neuronal excitability and the intrinsic plasticity of the Purkinje neurons as well as cerebellar memory consolidation.SIGNIFICANCE STATEMENT Stromal interaction molecule 1 (STIM1), which regulates the refilling of ER Ca2+, has been investigated in several systems including the CNS. In addition to a previous study showing that STIM1 regulates dendritic ER Ca2+ refilling and mGluR1-mediated synaptic transmission, we provide compelling evidence describing a novel role of STIM1 in spike firing Purkinje neurons. We found that STIM1 regulates cytosolic Ca2+ clearance of the soma during spike firing, and the interruption of this cytosolic Ca2+ clearing disrupts neuronal excitability and cerebellar memory consolidation. Our results provide new insights into neuronal functions of STIM1 from single neuronal Ca2+ dynamics to behavior level.

Sequestration of PRMT1 and Nd1-L mRNA into ALS-linked FUS mutant R521C-positive aggregates contributes to neurite degeneration upon oxidative stress.

Mutations in fused in sarcoma (FUS), a DNA/RNA binding protein, are associated with familial amyotrophic lateral sclerosis (ALS). However, little is known about how ALS-causing mutations alter protein-protein and protein-RNA complexes and contribute to neurodegeneration. In this study, we identified protein arginine methyltransferase 1 (PRMT1) as a protein that more avidly associates with ALS-linked FUS-R521C than with FUS-WT (wild type) or FUS-P525L using co-immunoprecipitation and LC-MS analysis. Abnormal association between FUS-R521C and PRMT1 requires RNA, but not methyltransferase activity. PRMT1 was sequestered into cytosolic FUS-R521C-positive stress granule aggregates. Overexpression of PRMT1 rescued neurite degeneration caused by FUS-R521C upon oxidative stress, while loss of PRMT1 further accumulated FUS-positive aggregates and enhanced neurite degeneration. Furthermore, the mRNA of Nd1-L, an actin-stabilizing protein, was sequestered into the FUS-R521C/PRMT1 complex. Nd1-L overexpression rescued neurite shortening caused by FUS-R521C upon oxidative stress, while loss of Nd1-L further exacerbated neurite shortening. Altogether, these data suggest that the abnormal stable complex of FUS-R521C/PRMT1/Nd1-L mRNA could contribute to neurodegeneration upon oxidative stress. Overall, our study provides a novel pathogenic mechanism of the FUS mutation associated with abnormal protein-RNA complexes upon oxidative stress in ALS and provides insight into possible therapeutic targets for this pathology.

Enhancing inhibitory synaptic function reverses spatial memory deficits in Shank2 mutant mice.

Autism spectrum disorders (ASDs) are a group of developmental disorders that cause variable and heterogeneous phenotypes across three behavioral domains such as atypical social behavior, disrupted communications, and highly restricted and repetitive behaviors. In addition to these core symptoms, other neurological abnormalities are associated with ASD, including intellectual disability (ID). However, the molecular etiology underlying these behavioral heterogeneities in ASD is unclear. Mutations in SHANK2 genes are associated with ASD and ID. Interestingly, two lines of Shank2 knockout mice (e6-7 KO and e7 KO) showed shared and distinct phenotypes. Here, we found that the expression levels of Gabra2, as well as of GABA receptor-mediated inhibitory neurotransmission, are reduced in Shank2 e6-7, but not in e7 KO mice compared with their own wild type littermates. Furthermore, treatment of Shank2 e6-7 KO mice with an allosteric modulator for the GABAA receptor reverses spatial memory deficits, indicating that reduced inhibitory neurotransmission may cause memory deficits in Shank2 e6-7 KO mice. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

Cell type-specific roles of RAS-MAPK signaling in learning and memory: Implications in neurodevelopmental disorders.

The RAS-mitogen-activated protein kinase (MAPK) signaling pathway plays critical roles in brain function, including learning and memory. Mutations of molecules in the RAS-MAPK pathway are associated with a group of disorders called RASopathies, which include Noonan syndrome, neurofibromatosis type 1, Costello syndrome, Noonan syndrome with multiple lentigines, Legius syndrome, and cardio-facio-cutaneous syndrome. RASopathies share certain clinical symptoms, including craniofacial abnormalities, heart defects, delayed growth, and cognitive deficits such as learning disabilities, while each individual syndrome also displays unique phenotypes. Recent studies using mouse models of RASopathies showed that each disorder may have a distinct molecular and cellular etiology depending on the cellular specificity of the mutated molecules. Here, we review the cell-type specific roles of the regulators of the RAS-MAPK pathway in cognitive function (learning and memory) and their contribution to the development of RASopathies. We also discussed recent technical advances in analyzing cell type-specific transcriptomes and proteomes in the nervous system. Understanding specific mechanisms for these similar but distinct disorders would facilitate the development of mechanism-based individualized treatment for RASopathies.

Impaired learning and memory in CD38 null mutant mice.

CD38 is an enzyme that catalyzes the formation of cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate, both of which are involved in the mobilization of Ca(2+) from intracellular stores. Recently, CD38 has been shown to regulate oxytocin release from hypothalamic neurons. Importantly, CD38 mutations are associated with autism spectrum disorders (ASD) and CD38 knockout (CD38(-/-)) mice display ASD-like behavioral phenotypes including deficient parental behavior and poor social recognition memory. Although ASD and learning deficits commonly co-occur, the role of CD38 in learning and memory has not been investigated. We report that CD38(-/-) mice show deficits in various learning and memory tasks such as the Morris water maze, contextual fear conditioning, and the object recognition test. However, either long-term potentiation or long-term depression is not impaired in the hippocampus of CD38(-/-) mice. Our results provide convincing evidence that CD38(-/-) mice show deficits in various learning and memory tasks including spatial and non-spatial memory tasks. Our data demonstrate that CD38 is critical for regulating hippocampus-dependent learning and memory without modulating synaptic plasticity.

Mechanism and treatment for learning and memory deficits in mouse models of Noonan syndrome.

In Noonan syndrome (NS) 30-50% of subjects show cognitive deficits of unknown etiology and with no known treatment. Here, we report that knock-in mice expressing either of two NS-associated mutations in Ptpn11, which encodes the nonreceptor protein tyrosine phosphatase Shp2, show hippocampal-dependent impairments in spatial learning and deficits in hippocampal long-term potentiation (LTP). In addition, viral overexpression of an NS-associated allele PTPN11(D61G) in adult mouse hippocampus results in increased baseline excitatory synaptic function and deficits in LTP and spatial learning, which can be reversed by a mitogen-activated protein kinase kinase (MEK) inhibitor. Furthermore, brief treatment with lovastatin reduces activation of the GTPase Ras-extracellular signal-related kinase (Erk) pathway in the brain and normalizes deficits in LTP and learning in adult Ptpn11(D61G/+) mice. Our results demonstrate that increased basal Erk activity and corresponding baseline increases in excitatory synaptic function are responsible for the LTP impairments and, consequently, the learning deficits in mouse models of NS. These data also suggest that lovastatin or MEK inhibitors may be useful for treating the cognitive deficits in NS.

Autophagy regulates amyotrophic lateral sclerosis-linked fused in sarcoma-positive stress granules in neurons.

Mutations in fused in sarcoma (FUS), a DNA/RNA binding protein, have been associated with familial amyotrophic lateral sclerosis (fALS), which is a fatal neurodegenerative disease that causes progressive muscular weakness and has overlapping clinical and pathologic characteristics with frontotemporal lobar degeneration. However, the role of autophagy in regulation of FUS-positive stress granules (SGs) and aggregates remains unclear. We found that the ALS-linked FUS(R521C) mutation causes accumulation of FUS-positive SGs under oxidative stress, leading to a disruption in the release of FUS from SGs in cultured neurons. Autophagy controls the quality of proteins or organelles; therefore, we checked whether autophagy regulates FUS(R521C)-positive SGs. Interestingly, FUS(R521C)-positive SGs were colocalized to RFP-LC3-positive autophagosomes. Furthermore, FUS-positive SGs accumulated in atg5(-/-) mouse embryonic fibroblasts (MEFs) and in autophagy-deficient neurons. However, FUS(R521C) expression did not significantly impair autophagic degradation. Moreover, autophagy activation with rapamycin reduced the accumulation of FUS-positive SGs in an autophagy-dependent manner. Rapamycin further reduced neurite fragmentation and cell death in neurons expressing mutant FUS under oxidative stress. Overall, we provide a novel pathogenic mechanism of ALS associated with a FUS mutation under oxidative stress, as well as therapeutic insight regarding FUS pathology associated with excessive SGs.

Autophagy negatively regulates early axon growth in cortical neurons.

Neurite growth requires neurite extension and retraction, which are associated with protein degradation. Autophagy is a conserved bulk degradation pathway that regulates several cellular processes. However, little is known about autophagic regulation during early neurite growth. In this study, we investigated whether autophagy was involved in early neurite growth and how it regulated neurite growth in primary cortical neurons. Components of autophagy were expressed and autophagy was activated during early neurite growth. Interestingly, inhibition of autophagy by atg7 small interfering RNA (siRNA) caused elongation of axons, while activation of autophagy by rapamycin suppressed axon growth. Surprisingly, inhibition of autophagy reduced the protein level of RhoA. Moreover, expression of RhoA suppressed axon overelongation mediated by autophagy inhibition, whereas inhibition of the RhoA signaling pathway by Y-27632 recovered rapamycin-mediated suppression of axon growth. Interestingly, hnRNP-Q1, which negatively regulates RhoA, accumulated in autophagy-deficient neurons, while its protein level was reduced by autophagy activation. Overall, our study suggests that autophagy negatively regulates axon extension via the RhoA-ROCK pathway by regulating hnRNP-Q1 in primary cortical neurons. Therefore, autophagy might serve as a fine-tuning mechanism to regulate early axon extension.

ALS/FTLD-linked TDP-43 regulates neurite morphology and cell survival in differentiated neurons.

Tar-DNA binding protein of 43kDa (TDP-43) has been characterized as a major component of protein aggregates in brains with neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). However, physiological roles of TDP-43 and early cellular pathogenic effects caused by disease associated mutations in differentiated neurons are still largely unknown. Here, we investigated the physiological roles of TDP-43 and the effects of missense mutations associated with diseases in differentiated cortical neurons. The reduction of TDP-43 by siRNA increased abnormal neurites and decreased cell viability. ALS/FTLD-associated missense mutant proteins (A315T, Q331K, and M337V) were partially mislocalized to the cytosol and neurites when compared to wild-type and showed abnormal neurites similar to those observed in cases of loss of TDP-43. Interestingly, cytosolic expression of wild-type TDP-43 with mutated nuclear localization signals also induced abnormal neurtie morphology and reduction of cell viability. However, there was no significant difference in the effects of cytosolic expression in neuronal morphology and cell toxicity between wild-type and missense mutant proteins. Thus, our results suggest that mislocalization of missense mutant TDP-43 may contribute to loss of TDP-43 function and affect neuronal morphology, probably via dominant negative action before severe neurodegeneration in differentiated cortical neurons.

The functional analysis of the CHMP2B missense mutation associated with neurodegenerative diseases in the endo-lysosomal pathway.

Endosomal sorting complexes required for transport (ESCRTs) regulate a key sorting step of protein trafficking between endosomal compartments in lysosomal degradation. Interestingly, mutations in charged multivesicular body protein 2B (CHMP2B), which is a core subunit of ESCRT-III, have been identified in some neurodegenerative diseases. However, the cellular pathogenesis resulting from CHMP2B missense mutations is unclear. Furthermore, little is known about their functional analysis in post-mitotic neurons. In order to examine their cellular pathogenesis, we analyzed their effects in the endo-lysosomal pathway in post-mitotic neurons. Interestingly, of the missense mutant proteins, CHMP2B(T104N) mostly accumulated in the Rab5- and Rab7-positive endosomes and caused delayed degradation of EGFR as compared to CHMP2B(WT). Furthermore, CHMP2B(T104N) showed less association with Vps4 ATPase and was avidly associated with Snf7-2, a core component of ESCRT-III, suggesting that it may cause defects in the process of dissociation from ESCRT. Of the missense variants, CHMP2B(T104N) caused prominent accumulation of autophagosomes. However, neuronal cell survival was not dramatically affected by expression of CHMP2B(T104N). These findings suggested that, from among the various missense mutants, CHMP2B(T104N) was associated with relatively mild cellular pathogenesis in post-mitotic neurons. This study provided a better understanding of the cellular pathogenesis of neurodegenerative diseases associated with various missense mutations of CHMP2B as well as endocytic defects.