LAMTOR1 inhibition of TRPML1-dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function.

Lysosomes function not only as degradatory compartments but also as dynamic intracellular calcium ion stores. The transient receptor potential mucolipin 1 (TRPML1) channel mediates lysosomal Ca2+ release, thereby participating in multiple cellular functions. The pentameric Ragulator complex, which plays a critical role in the activation of mTORC1, is also involved in lysosomal trafficking and is anchored to lysosomes through its LAMTOR1 subunit. Here, we report that the Ragulator restricts lysosomal trafficking in dendrites of hippocampal neurons via LAMTOR1-mediated tonic inhibition of TRPML1 activity, independently of mTORC1. LAMTOR1 directly interacts with TRPML1 through its N-terminal domain. Eliminating this inhibition in hippocampal neurons by LAMTOR1 deletion or by disrupting LAMTOR1-TRPML1 binding increases TRPML1-mediated Ca2+ release and facilitates dendritic lysosomal trafficking powered by dynein. LAMTOR1 deletion in the hippocampal CA1 region of adult mice results in alterations in synaptic plasticity, and in impaired object-recognition memory and contextual fear conditioning, due to TRPML1 activation. Mechanistically, changes in synaptic plasticity are associated with increased GluA1 dephosphorylation by calcineurin and lysosomal degradation. Thus, LAMTOR1-mediated inhibition of TRPML1 is critical for regulating dendritic lysosomal motility, synaptic plasticity, and learning.

Lack of UBE3A-Mediated Regulation of Synaptic SK2 Channels Contributes to Learning and Memory Impairment in the Female Mouse Model of Angelman Syndrome.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by severe developmental delay, motor impairment, language and cognition deficits, and often with increased seizure activity. AS is caused by deficiency of UBE3A, which is both an E3 ligase and a cofactor for transcriptional regulation. We previously showed that the small conductance potassium channel protein SK2 is a UBE3A substrate, and that increased synaptic SK2 levels contribute to impairments in synaptic plasticity and fear-conditioning memory, as inhibition of SK2 channels significantly improved both synaptic plasticity and fear memory in male AS mice. In the present study, we investigated UBE3a-mediated regulation of synaptic plasticity and fear-conditioning in female AS mice. Results from both western blot and immunofluorescence analyses showed that synaptic SK2 levels were significantly increased in hippocampus of female AS mice, as compared to wild-type (WT) littermates. Like in male AS mice, long-term potentiation (LTP) was significantly reduced while long-term depression (LTD) was enhanced at hippocampal CA3-CA1 synapses of female AS mice, as compared to female WT mice. Both alterations were significantly reduced by treatment with the SK2 inhibitor, apamin. The shunting effect of SK2 channels on NMDA receptor was significantly larger in female AS mice as compared to female WT mice. Female AS mice also showed impairment in both contextual and tone memory recall, and this impairment was significantly reduced by apamin treatment. Our results indicate that like male AS mice, female AS mice showed significant impairment in both synaptic plasticity and fear-conditioning memory due to increased levels of synaptic SK2 channels. Any therapeutic strategy to reduce SK2-mediated inhibition of NMDAR should be beneficial to both male and female patients.

Calpain-2 activation in mouse hippocampus plays a critical role in seizure-induced neuropathology.

Calpain has been proposed to play a critical role in the development of epilepsy. Here we used conditional calpain-2 knock-out (C2CKO) mice in a C57/Bl6 background and a selective calpain-2 inhibitor to analyze the role of calpain-2 in epilepsy. Neurodegeneration was evident in various hippocampal subfields, in particular in mossy cells in the hilus of the dentate gyrus (DG) in C57/Bl6 mice 7 days after kainic acid (KA)-induced seizures. Calpain-2 activation was still observed in mossy cells 7 days after seizures. Calpain activation, astroglial and microglial activation, neurodegeneration, and cognitive impairment were absent in C2CKO mice and in C57/Bl6 mice treated with a selective calpain-2 inhibitor for 7 days after seizure initiation. Levels of the potassium chloride cotransporter 2 (KCC2) were decreased in mossy cells 7 days after seizures and this decrease was prevented by calpain-2 deletion or selective inhibition. Our results indicate that prolonged calpain-2 activation plays a critical role in neuropathology following seizures. A selective calpain-2 inhibitor could represent a therapeutic treatment for seizure-induced neuropathology.

Impaired cerebellar plasticity and eye-blink conditioning in calpain-1 knock-out mice.

Calpain-1 and calpain-2 are involved in the regulation of several signaling pathways and neuronal functions in the brain. Our recent studies indicate that calpain-1 is required for hippocampal synaptic plasticity, including long-term depression (LTD) and long-term potentiation (LTP) in field CA1. However, little is known regarding the contributions of calpain-1 to cerebellar synaptic plasticity. Low frequency stimulation (LFS, 5 Hz, 5 min)-induced LTP at parallel fibers to Purkinje cell synapses was markedly impaired in cerebellar slices from calpain-1 knock-out (KO) mice. Application of a selective calpain-2 inhibitor enhanced LFS-induced LTP in both wild-type (WT) and calpain-1 KO mice. Three protocols were used to induce LTD at these synapses: LFS (1 Hz, 15 min), perfusion with high potassium and glutamate (K-Glu) or dihydroxyphenylglycine (DHPG), a mGluR1 agonist. All three forms of LTD were impaired in calpain-1 KO mice. DHPG application stimulated calpain-1 but not calpain-2 in cerebellar slices, and DHPG-induced LTD impairment was reversed by application of a protein phosphatase 2A (PP2A) inhibitor, okadaic acid. As in hippocampus, BDNF induced calpain-1 activation and PH domain and Leucine-rich repeat Protein Phosphatase 1/suprachiasmatic nucleus oscillatory protein (PHLPP1/SCOP) degradation followed by extracellular signal-regulated kinase (ERK) activation, as well as calpain-2 activation leading to degradation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) in cerebellar slices. The role of calpain-1 in associative learning was evaluated in the delay eyeblink conditioning (EBC). Calpain-1 KO mice exhibited significant learning impairment in EBC during the first 2 days of acquisition training. However, after 5 days of training, the percentage of conditioned responses (CRs) between calpain-1 KO and WT mice was identical. Both calpain-1 KO and WT mice exhibited typical extinction patterns. Our results indicate that calpain-1 plays critical roles in multiple forms of synaptic plasticity and associative learning in both hippocampus and cerebellum.

mTORC1-S6K1 inhibition or mTORC2 activation improves hippocampal synaptic plasticity and learning in Angelman syndrome mice.

Emerging evidence is implicating abnormal activation of the mechanistic target of rapamycin (mTOR) pathway in several monogenetic neuropsychiatric disorders, including Angelman syndrome (AS), which is caused by deficiency in maternally inherited UBE3A. Using an AS mouse model, we show that semi-chronic rapamycin treatment improves long-term potentiation (LTP) and actin polymerization in hippocampal slices, spine morphology, and fear-conditioning learning. Activity of mTORC1 and of its downstream substrate, S6K1, was increased in hippocampus of AS mice. However, mTORC2 activity, as reflected by PKCα levels, was decreased. Both increased mTORC1 and decreased mTORC2 activities were reversed by semi-chronic rapamycin treatment. Acute treatment of hippocampal slices from AS mice with rapamycin or an S6K1 inhibitor, PF4708671, improved LTP, restored actin polymerization, and normalized mTORC1 and mTORC2 activity. These treatments also reduced Arc levels in AS mice. Treatment with Torin 1, an inhibitor of both mTORC1 and mTORC2, partially rescued LTP and actin polymerization in hippocampal slices from AS mice, while partially impairing them in wild-type (WT) mice. Torin 1 decreased mTORC1 and increased mTORC2 activity in slices from AS mice but inhibited both mTORC1 and mTORC2 in WT mice. Finally, an mTORC2 activator, A-443654, increased hippocampal LTP in AS mice and actin polymerization in both WT and AS mice. Collectively, these results indicate that events set in motion by increased mTORC1 and decreased mTORC2 activities, including increased Arc translation and impaired actin remodeling, are crucial in AS pathogenesis. Therefore, selectively targeting these two master kinase complexes may provide new therapeutic approaches for AS treatment.

Deleting both PHLPP1 and CANP1 rescues impairments in long-term potentiation and learning in both single knockout mice.

Calpain-1 (CANP1) has been shown to play a critical role in synaptic plasticity and learning and memory, as its deletion in mice results in impairment in theta-burst stimulation- (TBS) induced LTP and various forms of learning and memory. Likewise, PHLPP1 (aka SCOP) has also been found to participate in learning and memory, as PHLPP1 overexpression impairs hippocampus-dependent learning. We previously showed that TBS-induced LTP was associated with calpain-1 mediated truncation of PHLPP1.To better understand the roles of these 2 genes in synaptic plasticity and learning and memory, we generated a double knockout (DKO) mouse by crossing the parent strains. Surprisingly, DKO mice exhibit normal TBS-induced LTP, and the learning impairments in fear conditioning and novel object or novel location recognition were absent in the DKO mice. Moreover, TBS-induced ERK activation in field CA1 of hippocampal slices, which is impaired in both single deletion mice, was restored in the DKO mice. These results further strengthen the roles of both CANP1 and PHLPP1 in synaptic plasticity and learning and memory, and illustrate the complexities of the interactions between multiple pathways participating in synaptic plasticity.

Different patterns of electrical activity lead to long-term potentiation by activating different intracellular pathways.

Deciphering and storing information coded in different firing patterns are important properties of neuronal networks, as they allow organisms to respond and adapt to external and internal events. Here we report that hippocampal CA1 pyramidal neurons respond to brief bursts of high-frequency stimulation (HFS) and θ burst stimulation (TBS) with long-lasting enhanced responses (long-term potentiation [LTP]), albeit by engaging different signaling pathways. TBS induces LTP through calpain-1-mediated suprachiasmatic nucleus circadian oscillatory protein degradation, ERK activation, and actin polymerization, whereas HFS requires adenosine A2 receptors, PKA, and actin polymerization. TBS- but not HFS-induced LTP is impaired in calpain-1 knock-out mice. However, TBS-induced LTP and learning impairment in knock-out mice are restored by activating the HFS pathway. Thus, different patterns of rhythmic activities trigger potentiation by activating different pathways, and cross talks between these can be used to restore LTP and learning when elements of the pathways are impaired.

Imbalanced mechanistic target of rapamycin C1 and C2 activity in the cerebellum of Angelman syndrome mice impairs motor function.

Angelman syndrome (AS) is a neurogenetic disorder caused by deficiency of maternally expressed ubiquitin-protein ligase E3A (UBE3A), an E3 ligase that targets specific proteins for proteasomal degradation. Although motor function impairment occurs in all patients with AS, very little research has been done to understand and treat it. The present study focuses on Ube3A deficiency-induced alterations in signaling through the mechanistic target of rapamycin (mTOR) pathway in the cerebellum of the AS mouse model and on potential therapeutic applications of rapamycin. Levels of tuberous sclerosis complex 2 (TSC2), a negative regulator of mTOR, were increased in AS mice compared with wild-type mice; however, TSC2 inhibitory phosphorylation was also increased. Correspondingly, levels of phosphorylated/active mTOR were increased. Phosphorylation of the mTORC1 substrates S6 kinase 1 (S6K1) and S6 was elevated, whereas that of the mTORC2 substrates AKT and N-myc downstream regulated 1 was decreased, suggesting enhanced mTORC1 but inhibited mTORC2 signaling. Semi-chronic treatment of AS mice with rapamycin not only improved their motor performance but also normalized mTORC1 and mTORC2 signaling. Furthermore, inhibitory phosphorylation of rictor, a key regulatory/structural subunit of the mTORC2 complex, was increased in AS mice and decreased after rapamycin treatment. These results indicate that Ube3A deficiency leads to overactivation of the mTORC1-S6K1 pathway, which in turn inhibits rictor, resulting in decreased mTORC2 signaling in Purkinje neurons of AS mice. Finally, rapamycin treatment also improved dendritic spine morphology in AS mice, through inhibiting mTORC1 and possibly enhancing mTORC2-mediated regulation of synaptic cytoskeletal elements. Collectively, our results indicate that the imbalance between mTORC1 and mTORC2 activity may contribute to synaptic pathology and motor impairment in AS.

Activity-dependent rapid local RhoA synthesis is required for hippocampal synaptic plasticity.

Dendritic protein synthesis and actin cytoskeleton reorganization are important events required for the consolidation of hippocampal LTP and memory. However, the temporal and spatial relationships between these two processes remain unclear. Here, we report that treatment of adult rat hippocampal slices with BDNF or with tetraethylammonium (TEA), which induces a chemical form of LTP, produces a rapid and transient increase in RhoA protein levels. Changes in RhoA were restricted to dendritic spines of CA3 and CA1 and require de novo protein synthesis regulated by mammalian target of rapamycin (mTOR). BDNF-mediated stimulation of RhoA activity, cofilin phosphorylation, and actin polymerization were completely suppressed by protein synthesis inhibitors. Furthermore, intrahippocampal injections of RhoA antisense oligodeoxynucleotides inhibited theta burst stimulation (TBS)-induced RhoA upregulation in dendritic spines and prevented LTP consolidation. Addition of calpain inhibitors after BDNF or TEA treatment maintained RhoA levels elevated and prolonged the effects of BDNF and TEA on actin polymerization. Finally, the use of isoform-selective calpain inhibitors revealed that calpain-2 was involved in RhoA synthesis, whereas calpain-1 mediated RhoA degradation. Overall, this mechanism provides a novel link between dendritic protein synthesis and reorganization of the actin cytoskeleton in hippocampal dendritic spines during LTP consolidation.

Calpain-1 and calpain-2 play opposite roles in retinal ganglion cell degeneration induced by retinal ischemia/reperfusion injury.

Calpain has been shown to be involved in neurodegeneration, and in particular in retinal ganglion cell (RGC) death resulting from increased intraocular pressure (IOP) and ischemia. However, the specific roles of the two major calpain isoforms, calpain-1 and calpain-2, in RGC death have not been investigated. Here, we show that calpain-1 and calpain-2 were sequentially activated in RGC dendrites after acute IOP elevation. By combining the use of a selective calpain-2 inhibitor (C2I) and calpain-1 KO mice, we demonstrated that calpain-1 activity supported survival, while calpain-2 activity promoted cell death of RGCs after IOP elevation. Calpain-1 activation cleaved PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) and activated the Akt pro-survival pathway, while calpain-2 activation cleaved striatal-enriched protein tyrosine phosphatase (STEP) and activated STEP-mediated pro-death pathway in RGCs after IOP elevation. Systemic or intravitreal C2I injection to wild-type mice 2h after IOP elevation promoted RGC survival and improved visual function. Our data indicate that calpain-1 and calpain-2 play opposite roles in high IOP-induced ischemic injury and that a selective calpain-2 inhibitor could prevent acute glaucoma-induced RGC death and blindness.

Serine/threonine kinases 31(STK31) may be a novel cellular target gene for the HPV16 oncogene E7 with potential as a DNA hypomethylation biomarker in cervical cancer.

BACKGROUND: Cervical cancer (CC) is a leading cause of mortality in females, especially in developing countries. The two viral oncoproteins E6 and E7 mediate the oncogenic activities of high-risk human papillomavirus (hrHPV), and hrHPV, especially HPV16 or/and HPV18 (HPV16/18) play critical roles in CC through different pathways. STK31 gene of which the expression has been proven to be regulated by the methylation status of its promoter, is one of the novel cancer/testis (CT) genes and plays important roles in human cancers. Reasearches have indicated that viral infection is correlated to the methylation statuses of some genes. Herein, we detected methylation status of the STK31 gene in cervical tumors and explored its interaction with HPV16 or/and HPV18 (HPV16/18) infection. METHODS: Bisulfite genomic sequencing PCR (BGS) combined with TA clone, methylation-specific PCR (MSP) were used to analyze methylation statuses of the STK31 gene promoter/exon 1 region in HPV16/18-positive, HPV-negative CC cell lines; ectopically expressed HPV16 E6, -E7, and -E6/E7 CC cells; normal cervical tissues and cervical tumor tissues of different stages. The mRNA and protein expressions of STK31 were detected by RT-PCR and western blotting. RESULTS: The STK31 gene promoter/exon 1 was hypomethylated in the HPV16/18-positive cell lines HeLa, SiHa and CaSki, and the mRNA and protein expression were detected. In contrast, the STK31 gene exhibited hypermethylation and silenced expression in the HPV-negative CC cells C33A and HT-3. Compared with the primary HPV-negative CC cell lines, the STK31 methylation was downregulated, and STK31 expression was induced in the HPV16E7/E67 transfected cells. The methylation statuses and expressions of STK31 were verified in the cervical tumor samples at different stages. Additionally, chemotherapy treatment may influence STK31 expression by regulating its methylation status. CONCLUSIONS: STK31 may be a novel cellular target gene for the HPV16 oncogeneE7. The HPV16 oncogene E7 may affect STK31 expression through a methylation-mediated mechanism. The aberrant methylation of the STK31 promoter/exon 1 region may be a precursor of human cervical carcinogenesis and a potential DNA aberrant methylation biomarker of conditions ranging from precancerous disease to invasive cancer.

Distinct roles for µ-calpain and m-calpain in synaptic NMDAR-mediated neuroprotection and extrasynaptic NMDAR-mediated neurodegeneration.

Prolonged calpain activation is widely recognized as a key component of neurodegeneration in a variety of pathological conditions. Numerous reports have also indicated that synaptic activation of NMDA receptors (NMDARs) provides neuroprotection against a variety of insults. Here, we report the paradoxical finding that such neuroprotection involves calpain activation. NMDAR activation in cultured rat cortical neurons was neuroprotective against starvation and oxidative stress-induced damage. It also resulted in the degradation of two splice variants of PH domain and Leucine-rich repeat Protein Phosphatase 1 (PHLPP1), PHLPP1α and PHLPP1ß, which inhibit the Akt and ERK1/2 pathways. Synaptic NMDAR-induced neuroprotection and PHLPP1 degradation were blocked by calpain inhibition. Lentiviral knockdown of PHLPP1 mimicked the neuroprotective effects of synaptic NMDAR activation and occluded the effects of calpain inhibition on neuroprotection. In contrast to synaptic NMDAR activation, extrasynaptic NMDAR activation had no effect on PHLPP1 and the Akt and ERK1/2 pathways, but resulted in calpain-mediated degradation of striatal-enriched protein tyrosine phosphatase (STEP) and neuronal death. Using µ-calpain- and m-calpain-selective inhibitors and µ-calpain and m-calpain siRNAs, we found that µ-calpain-dependent PHLPP1 cleavage was involved in synaptic NMDAR-mediated neuroprotection, while m-calpain-mediated STEP degradation was associated with extrasynaptic NMDAR-induced neurotoxicity. Furthermore, m-calpain inhibition reduced while µ-calpain knockout exacerbated NMDA-induced neurotoxicity in acute mouse hippocampal slices. Thus, synaptic NMDAR-coupled µ-calpain activation is neuroprotective, while extrasynaptic NMDAR-coupled m-calpain activation is neurodegenerative. These results help to reconcile a number of contradictory results in the literature and have critical implications for the understanding and potential treatment of neurodegenerative diseases.

Calpain-2-mediated PTEN degradation contributes to BDNF-induced stimulation of dendritic protein synthesis.

Memory consolidation has been suggested to be protein synthesis dependent. Previous data indicate that BDNF-induced dendritic protein synthesis is a key event in memory formation through activation of the mammalian target of rapamycin (mTOR) pathway. BDNF also activates calpain, a calcium-dependent cysteine protease, which has been shown to play a critical role in learning and memory. This study was therefore directed at testing the hypothesis that calpain activity is required for BDNF-stimulated local protein synthesis, and at identifying the underlying molecular mechanism. In rat hippocampal slices, cortical synaptoneurosomes, and cultured neurons, BDNF-induced mTOR pathway activation and protein translation were blocked by calpain inhibition. BDNF treatment rapidly reduced levels of hamartin and tuberin, negative regulators of mTOR, in a calpain-dependent manner. Treatment of brain homogenates with purified calpain-1 and calpain-2 truncated both proteins. BDNF treatment increased phosphorylation of both Akt and ERK, but only the effect on Akt was blocked by calpain inhibition. Levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a phosphatase that inactivates Akt, were decreased following BDNF treatment, and calpain inhibition reversed this effect. Calpain-2, but not calpain-1, treatment of brain homogenates resulted in PTEN degradation. In cultured cortical neurons, knockdown of calpain-2, but not calpain-1, by small interfering RNA completely suppressed the effect of BDNF on mTOR activation. Our results reveal a critical role for calpain-2 in BDNF-induced mTOR signaling and dendritic protein synthesis via PTEN, hamartin, and tuberin degradation. This mechanism therefore provides a link between proteolysis and protein synthesis that might contribute to synaptic plasticity.

Revisiting the calpain hypothesis of learning and memory 40 years later.

In 1984, Gary Lynch and Michel Baudry published in Science a novel biochemical hypothesis for learning and memory, in which they postulated that the calcium-dependent protease, calpain, played a critical role in regulating synaptic properties and the distribution of glutamate receptors, thereby participating in memory formation in hippocampus. Over the following 40 years, much work has been done to refine this hypothesis and to provide convincing arguments supporting what was viewed at the time as a simplistic view of synaptic biochemistry. We have now demonstrated that the two major calpain isoforms in the brain, calpain-1 and calpain-2, execute opposite functions in both synaptic plasticity/learning and memory and in neuroprotection/neurodegeneration. Thus, calpain-1 activation is required for triggering long-term potentiation (LTP) of synaptic transmission and learning of episodic memory, while calpain-2 activation limits the magnitude of LTP and the extent of learning. On the other hand, calpain-1 is neuroprotective while calpain-2 is neurodegenerative, and its prolonged activation following various types of brain insults leads to neurodegeneration. The signaling pathways responsible for these functions have been identified and involve local protein synthesis, cytoskeletal regulation, and regulation of glutamate receptors. Human families with mutations in calpain-1 have been reported to have impairment in motor and cognitive functions. Selective calpain-2 inhibitors have been synthesized and clinical studies to test their potential use to treat disorders associated with acute neuronal damage, such as traumatic brain injury, are being planned. This review will illustrate the long and difficult journey to validate a bold hypothesis.

Identification and neuroprotective properties of NA-184, a calpain-2 inhibitor.

Our laboratory has shown that calpain-2 activation in the brain following acute injury is directly related to neuronal damage and the long-term functional consequences of the injury, while calpain-1 activation is generally neuroprotective and calpain-1 deletion exacerbates neuronal injury. We have also shown that a relatively selective calpain-2 inhibitor, referred to as C2I, enhanced long-term potentiation and learning and memory, and provided neuroprotection in the controlled cortical impact (CCI) model of traumatic brain injury (TBI) in mice. Using molecular dynamic simulation and Site Identification by Ligand Competitive Saturation (SILCS) software, we generated about 130 analogs of C2I and tested them in a number of in vitro and in vivo assays. These led to the identification of two interesting compounds, NA-112 and NA-184. Further analyses indicated that NA-184, (S)-2-(3-benzylureido)-N-((R,S)-1-((3-chloro-2-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide, selectively and dose-dependent inhibited calpain-2 activity without evident inhibition of calpain-1 at the tested concentrations in mouse brain tissues and human cell lines. Like NA-112, NA-184 inhibited TBI-induced calpain-2 activation and cell death in mice and rats, both male and females. Pharmacokinetic and pharmacodynamic analyses indicated that NA-184 exhibited properties, including stability in plasma and liver and blood-brain barrier permeability, that make it a good clinical candidate for the treatment of TBI.

Learning and memory: an emergent property of cell motility.

In this review, we develop the argument that the molecular/cellular mechanisms underlying learning and memory are an adaptation of the mechanisms used by all cells to regulate cell motility. Neuronal plasticity and more specifically synaptic plasticity are widely recognized as the processes by which information is stored in neuronal networks engaged during the acquisition of information. Evidence accumulated over the last 25 years regarding the molecular events underlying synaptic plasticity at excitatory synapses has shown the remarkable convergence between those events and those taking place in cells undergoing migration in response to extracellular signals. We further develop the thesis that the calcium-dependent protease, calpain, which we postulated over 25 years ago to play a critical role in learning and memory, plays a central role in the regulation of both cell motility and synaptic plasticity. The findings discussed in this review illustrate the general principle that fundamental cell biological processes are used for a wide range of functions at the level of organisms.

Role of calpain-mediated p53 truncation in semaphorin 3A-induced axonal growth regulation.

Neurite outgrowth represents a critical stage in the correct development of neuronal circuitries, and is dependent on the complex regulation of actin filament and microtubule dynamics by intrinsic as well as extrinsic signals. Previous studies have implicated the tumor suppressor factor, p53, in the regulation of axonal outgrowth through a nontranscriptional effect involving local regulation of the Rho kinase signaling pathway that controls these dynamics. In the present study, we first showed that semaphorin 3A-induced growth cone collapse in cultured hippocampal neurons was associated with the partial truncation of phosphorylated p53, and that both effects were prevented by calpain inhibition with either m-calpain-specific siRNA or inhibitors. We further determined that semaphorin 3A-mediated calpain activation and growth cone collapse were associated with m-calpain phosphorylation and prevented by inhibition of MAPK, ERK, or p38. In vitro studies confirmed that p53 and especially phosphorylated p53 were partially truncated by calpain. Thus, our results indicate that semaphorin 3A-mediated growth cone collapse is mediated in part by m-calpain activation, possibly through MAPK-mediated phosphorylation, and the resulting truncation of phosphorylated p53, leading to Rho kinase activation and cytoskeletal reorganization. They provide a pathway by which extrinsic signals regulate axonal growth through activation of m-calpain and p53 truncation.

Calpain-2 Inhibitors as Therapy for Traumatic Brain Injury.

While calpains have long been implicated in neurodegeneration, no calpain inhibitor has been developed for the treatment of neurodegeneration. This is partly due to the lack of understanding of the specific functions of most of the 15 members of the calpain family. Work from our laboratory over the last 5-10 years has revealed that calpain-1 and calpain-2, two of the major calpain isoforms in the brain, play opposite roles in both synaptic plasticity/learning and memory and neuroprotection/neurodegeneration. Thus, calpain-1 activation is required for triggering certain forms of synaptic plasticity and for learning some types of information and is neuroprotective. In contrast, calpain-2 activation limits the extent of synaptic plasticity and of learning and is neurodegenerative. These results have been validated with the use of calpain-1 knock-out mice and mice with a selective calpain-2 deletion in excitatory neurons of the forebrain. Through a medicinal chemistry campaign, we have identified a number of selective calpain-2 inhibitors and shown that these inhibitors do facilitate learning of certain tasks and are neuroprotective in a number of animal models of acute neurodegeneration. One of these inhibitors, NA-184, is currently being developed for the treatment of traumatic brain injury, and clinical trials are being planned.

UBE3A deficiency-induced autophagy is associated with activation of AMPK-ULK1 and p53 pathways.

Angelman Syndrome (AS) is a neurodevelopmental disorder caused by deficiency of the maternally expressed UBE3A gene. The UBE3A proteins functions both as an E3 ligase in the ubiquitin-proteasome system (UPS), and as a transcriptional co-activator for steroid hormone receptors. Here we investigated the effects of UBE3A deficiency on autophagy in the cerebellum of AS mice and in COS1 cells. Numbers and size of LC3- and LAMP2-immunopositive puncta were increased in cerebellar Purkinje cells of AS mice, as compared to wildtype mice. Western blot analysis showed an increase in the conversion of LC3I to LC3II in AS mice, as expected from increased autophagy. Levels of active AMPK and of one of its substrates, ULK1, a factor involved in autophagy initiation, were also increased. Colocalization of LC3 with LAMP2 was increased and p62 levels were decreased, indicating an increase in autophagy flux. UBE3A deficiency was also associated with reduced levels of phosphorylated p53 in the cytosol and increased levels in nuclei, which favors autophagy induction. UBE3A siRNA knockdown in COS-1 cells resulted in increased size and intensity of LC3-immunopositive puncta and increased the LC3 II/I ratio, as compared to control siRNA-treated cells, confirming the results found in the cerebellum of AS mice. These results indicate that UBE3A deficiency enhances autophagic activity through activation of the AMPK-ULK1 pathway and alterations in p53.

Contextual fear memory impairment in Angelman syndrome model mice is associated with altered transcriptional responses.

Angelman syndrome (AS) is a rare neurogenetic disorder caused by UBE3A deficiency and characterized by severe developmental delay, cognitive impairment, and motor dysfunction. In the present study, we performed RNA-seq on hippocampal samples from both wildtype (WT) and AS male mice, with or without contextual fear memory recall. There were 281 recall-associated differentially expressed genes (DEGs) in WT mice and 268 DEGs in AS mice, with 129 shared by the two genotypes. Gene ontology analysis showed that extracellular matrix and stimulation-induced response genes were prominently enriched in recall-associated DEGs in WT mice, while nuclear acid metabolism and tissue development genes were highly enriched in those from AS mice. Further analyses showed that the 129 shared DEGs belonged to nuclear acid metabolism and tissue development genes. Unique recall DEGs in WT mice were enriched in biological processes critical for synaptic plasticity and learning and memory, including the extracellular matrix network clustered around fibronectin 1 and collagens. In contrast, AS-specific DEGs were not enriched in any known pathways. These results suggest that memory recall in AS mice, while altering the transcriptome, fails to recruit memory-associated transcriptional programs, which could be responsible for the memory impairment in AS mice.