The Regulation of the Disease-Causing Gene FXN.

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease caused in almost all patients by expanded guanine-adenine-adenine (GAA) trinucleotide repeats within intron 1 of the FXN gene. This results in a relative deficiency of frataxin, a small nucleus-encoded mitochondrial protein crucial for iron-sulfur cluster biogenesis. Currently, there is only one medication, omaveloxolone, available for FRDA patients, and it is limited to patients 16 years of age and older. This necessitates the development of new medications. Frataxin restoration is one of the main strategies in potential treatment options as it addresses the root cause of the disease. Comprehending the control of frataxin at the transcriptional, post-transcriptional, and post-translational stages could offer potential therapeutic approaches for addressing the illness. This review aims to provide a general overview of the regulation of frataxin and its implications for a possible therapeutic treatment of FRDA.

A peptide derived from TID1S rescues frataxin deficiency and mitochondrial defects in FRDA cellular models.

Friedreich's ataxia (FRDA), the most common recessive inherited ataxia, results from homozygous guanine-adenine-adenine (GAA) repeat expansions in intron 1 of the FXN gene, which leads to the deficiency of frataxin, a mitochondrial protein essential for iron-sulphur cluster synthesis. The study of frataxin protein regulation might yield new approaches for FRDA treatment. Here, we report tumorous imaginal disc 1 (TID1), a mitochondrial J-protein cochaperone, as a binding partner of frataxin that negatively controls frataxin protein levels. TID1 interacts with frataxin both in vivo in mouse cortex and in vitro in cortical neurons. Acute and subacute depletion of frataxin using RNA interference markedly increases TID1 protein levels in multiple cell types. In addition, TID1 overexpression significantly increases frataxin precursor but decreases intermediate and mature frataxin levels in HEK293 cells. In primary cultured human skin fibroblasts, overexpression of TID1S results in decreased levels of mature frataxin and increased fragmentation of mitochondria. This effect is mediated by the last 6 amino acids of TID1S as a peptide made from this sequence rescues frataxin deficiency and mitochondrial defects in FRDA patient-derived cells. Our findings show that TID1 negatively modulates frataxin levels, and thereby suggests a novel therapeutic target for treating FRDA.

A non-synonymous single nucleotide polymorphism in SIRT6 predicts neurological severity in Friedreich ataxia.

Introduction: Friedreich ataxia (FRDA) is a recessive neurodegenerative disease characterized by progressive ataxia, dyscoordination, and loss of vision. The variable length of the pathogenic GAA triplet repeat expansion in the FXN gene in part explains the interindividual variability in the severity of disease. The GAA repeat expansion leads to epigenetic silencing of FXN; therefore, variability in properties of epigenetic effector proteins could also regulate the severity of FRDA. Methods: In an exploratory analysis, DNA from 88 individuals with FRDA was analyzed to determine if any of five non-synonymous SNPs in HDACs/SIRTs predicted FRDA disease severity. Results suggested the need for a full analysis at the rs352493 locus in SIRT6 (p.Asn46Ser). In a cohort of 569 subjects with FRDA, disease features were compared between subjects homozygous for the common thymine SIRT6 variant (TT) and those with the less common cytosine variant on one allele and thymine on the other (CT). The biochemical properties of both variants of SIRT6 were analyzed and compared. Results: Linear regression in the exploratory cohort suggested that an SNP (rs352493) in SIRT6 correlated with neurological severity in FRDA. The follow-up analysis in a larger cohort agreed with the initial result that the genotype of SIRT6 at the locus rs352493 predicted the severity of disease features of FRDA. Those in the CT SIRT6 group performed better on measures of neurological and visual function over time than those in the more common TT SIRT6 group. The Asn to Ser amino acid change resulting from the SNP in SIRT6 did not alter the expression or enzymatic activity of SIRT6 or frataxin, but iPSC-derived neurons from people with FRDA in the CT SIRT6 group showed whole transcriptome differences compared to those in the TT SIRT6 group. Conclusion: People with FRDA in the CT SIRT6 group have less severe neurological and visual dysfunction than those in the TT SIRT6 group. Biochemical analyses indicate that the benefit conferred by T to C SNP in SIRT6 does not come from altered expression or enzymatic activity of SIRT6 or frataxin but is associated with changes in the transcriptome.

Frataxin controls ketone body metabolism through regulation of OXCT1.

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by the deficiency of mitochondrial protein frataxin, which plays a crucial role in iron-sulphur cluster formation and ATP production. The cellular function of frataxin is not entirely known. Here, we demonstrate that frataxin controls ketone body metabolism through regulation of 3-Oxoacid CoA-Transferase 1 (OXCT1), a rate limiting enzyme catalyzing the conversion of ketone bodies to acetoacetyl-CoA that is then fed into the Krebs cycle. Biochemical studies show a physical interaction between frataxin and OXCT1 both in vivo and in vitro. Frataxin overexpression also increases OXCT1 protein levels in human skin fibroblasts while frataxin deficiency decreases OXCT1 in multiple cell types including cerebellum and skeletal muscle both acutely and chronically, suggesting that frataxin directly regulates OXCT1. This regulation is mediated by frataxin-dependent suppression of ubiquitin-proteasome system (UPS)-dependent OXCT1 degradation. Concomitantly, plasma ketone bodies are significantly elevated in frataxin deficient knock-in/knockout (KIKO) mice with no change in the levels of other enzymes involved in ketone body production. In addition, ketone bodies fail to be metabolized to acetyl-CoA accompanied by increased succinyl-CoA in vitro in frataxin deficient cells, suggesting that ketone body elevation is caused by frataxin-dependent reduction of OXCT1 leading to deficits in tissue utilization of ketone bodies. Considering the potential role of metabolic abnormalities and deficiency of ATP production in FRDA, our results suggest a new role for frataxin in ketone body metabolism and also suggest modulation of OXCT1 may be a potential therapeutic approach for FRDA.

Clinical Evidence for Variegated Silencing in Patients With Friedreich Ataxia.

Background and Objectives: Friedreich ataxia (FRDA) is a neurodegenerative disease caused by a GAA triplet repeat (GAA-TR) expansion in intron 1 of the FXN gene. Patients have 100-1,300 GAA triplets compared with less than 30 in healthy controls. The GAA-TR expansion leads to FXN silencing, and consequent frataxin protein deficiency results in progressive ataxia, scoliosis, cardiomyopathy, and diabetes. The overt heterogeneity in age at onset and disease severity is explained partly by the length of the GAA-TR, in which shorter repeats correlate with milder disease. Evidence of variegated silencing in FRDA suggests that patients with shorter repeats retain a significant proportion of cells with FXN genes that have escaped GAA-TR expansion-induced silencing, explaining the less severe frataxin deficiency in this subpopulation. In ex vivo experiments, the proportion of spared cells negatively correlates with GAA-TR length until it plateaus at 500 triplets, an indication that the maximal number of silenced cells has been reached. In this study, we assessed whether an analogous ceiling effect occurs in severity of clinical features of FRDA by analyzing clinical outcome data. Methods: The FRDA Clinical Outcome Measures Study database was used for a cross-sectional analysis of 1,000 patients with FRDA. Frataxin levels were determined by lateral flow immunoassays. Results: The length of the GAA-TR in our cohort predicted frataxin level (R2 = 0.38, p < 0.0001) and age at onset (R2 = 0.46, p < 0.0001) but only with GAA-TRs with ≤700 triplets. Age and disease duration predicted performance on clinical outcome measures, and such predictions in linear regression models statistically improved in the subcohort of patients with >700 GAA triplets. The prevalence of cardiomyopathy and scoliosis increased as GAA-TR length increased up to 700 GAA triplets where prevalence plateaued. Discussion: Our data suggest that there is a ceiling effect on the clinical consequences of GAA-TR length in FRDA, as would be predicted by variegated silencing. Patients with GAA-TRs of >700 triplets represent a subgroup in which the severity of clinical manifestations based on GAA-TR length have reached maximal levels and therefore display limited clinical variability in disease progression.

Cerebellar Pathology in an Inducible Mouse Model of Friedreich Ataxia.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by deficiency of the mitochondrial protein frataxin. Lack of frataxin causes neuronal loss in various areas of the CNS and PNS. In particular, cerebellar neuropathology in FRDA patients includes loss of large principal neurons and synaptic terminals in the dentate nucleus (DN), and previous studies have demonstrated early synaptic deficits in the Knockin-Knockout mouse model of FRDA. However, the exact correlation of frataxin deficiency with cerebellar neuropathology remains unclear. Here we report that doxycycline-induced frataxin knockdown in a mouse model of FRDA (FRDAkd) leads to synaptic cerebellar degeneration that can be partially reversed by AAV8-mediated frataxin restoration. Loss of cerebellar Purkinje neurons and large DN principal neurons are observed in the FRDAkd mouse cerebellum. Levels of the climbing fiber-specific glutamatergic synaptic marker VGLUT2 decline starting at 4 weeks after dox induction, whereas levels of the parallel fiber-specific synaptic marker VGLUT1 are reduced by 18-weeks. These findings suggest initial selective degeneration of climbing fiber synapses followed by loss of parallel fiber synapses. The GABAergic synaptic marker GAD65 progressively declined during dox induction in FRDAkd mice, while GAD67 levels remained unaltered, suggesting specific roles for frataxin in maintaining cerebellar synaptic integrity and function during adulthood. Expression of frataxin following AAV8-mediated gene transfer partially restored VGLUT1/2 levels. Taken together, our findings show that frataxin knockdown leads to cerebellar degeneration in the FRDAkd mouse model, suggesting that frataxin helps maintain cerebellar structure and function.

Functional NMDA receptors are expressed by human pulmonary artery smooth muscle cells.

N-methyl-D-aspartate (NMDA) receptors are widely expressed in the central nervous system. However, their presence and function at extraneuronal sites is less well characterized. In the present study, we examined the expression of NMDA receptor subunit mRNA and protein in human pulmonary artery (HPA) by quantitative polymerase chain reaction (PCR), immunohistochemistry and immunoblotting. We demonstrate that both GluN1 and GluN2 subunit mRNAs are expressed in HPA. In addition, GluN1 and GluN2 (A-D) subunit proteins are expressed by human pulmonary artery smooth muscle cells (HPASMCs) in vitro and in vivo. These subunits localize on the surface of HPASMCs and form functional ion channels as evidenced by whole-cell patch-clamp electrophysiology and reduced phenylephrine-induced contractile responsiveness of human pulmonary artery by the NMDA receptor antagonist MK801 under hypoxic condition. HPASMCs also express high levels of serine racemase and vesicular glutamate transporter 1, suggesting a potential source of endogenous agonists for NMDA receptor activation. Our findings show HPASMCs express functional NMDA receptors in line with their effect on pulmonary vasoconstriction, and thereby suggest a novel therapeutic target for pharmacological modulations in settings associated with pulmonary vascular dysfunction.

Mitochondrial dysfunction in the development and progression of neurodegenerative diseases.

In addition to ATP synthesis, mitochondria are highly dynamic organelles that modulate apoptosis, ferroptosis, and inflammasome activation. Through executing these varied functions, the mitochondria play critical roles in the development and progression of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, and Friedreich ataxia, among others. Impaired mitochondrial biogenesis and abnormal mitochondrial dynamics contribute to mitochondrial dysfunction in these diseases. Additionally, dysfunctional mitochondria play critical roles in signaling for both inflammasome activation and ferroptosis. Therapeutics are being developed to circumvent inflammasome activation and ferroptosis in dysfunctional mitochondria. Targeting these aspects of mitochondrial dysfunction may present viable therapeutic strategies for combatting the neurodegenerative diseases. This review aims to summarize the role of the mitochondria in the development and progression of neurodegenerative diseases and to present current therapeutic approaches that target mitochondrial dysfunction in these diseases.

PPAR gamma agonist leriglitazone improves frataxin-loss impairments in cellular and animal models of Friedreich Ataxia.

Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is characterized by degeneration of the large sensory neurons and spinocerebellar tracts, cardiomyopathy, and increased incidence in diabetes. The underlying pathophysiological mechanism of FRDA, driven by a significantly decreased expression of frataxin (FXN), involves increased oxidative stress, reduced activity of enzymes containing iron­sulfur clusters (ISC), defective energy production, calcium dyshomeostasis, and impaired mitochondrial biogenesis, leading to mitochondrial dysfunction. The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcriptional factor playing a key role in mitochondrial function and biogenesis, fatty acid storage, energy metabolism, and antioxidant defence. It has been previously shown that the PPARγ/PPARγ coactivator 1 alpha (PGC-1α) pathway is dysregulated when there is frataxin deficiency, thus contributing to FRDA pathogenesis and supporting the PPARγ pathway as a potential therapeutic target. Here we assess whether MIN-102 (INN: leriglitazone), a novel brain penetrant and orally bioavailable PPARγ agonist with an improved profile for central nervous system (CNS) diseases, rescues phenotypic features in cellular and animal models of FRDA. In frataxin-deficient dorsal root ganglia (DRG) neurons, leriglitazone increased frataxin protein levels, reduced neurite degeneration and α-fodrin cleavage mediated by calpain and caspase 3, and increased survival. Leriglitazone also restored mitochondrial membrane potential and partially reversed decreased levels of mitochondrial Na+/Ca2+ exchanger (NCLX), resulting in an improvement of mitochondrial functions and calcium homeostasis. In frataxin-deficient primary neonatal cardiomyocytes, leriglitazone prevented lipid droplet accumulation without increases in frataxin levels. Furthermore, leriglitazone improved motor function deficit in YG8sR mice, a FRDA mouse model. In agreement with the role of PPARγ in mitochondrial biogenesis, leriglitazone significantly increased markers of mitochondrial biogenesis in FRDA patient cells. Overall, these results suggest that targeting the PPARγ pathway by leriglitazone may provide an efficacious therapy for FRDA increasing the mitochondrial function and biogenesis that could increase frataxin levels in compromised frataxin-deficient DRG neurons. Alternately, leriglitazone improved the energy metabolism by increasing the fatty acid ß-oxidation in frataxin-deficient cardiomyocytes without elevation of frataxin levels. This could be linked to a lack of significant mitochondrial biogenesis and cardiac hypertrophy. The results reinforced the different tissue requirement in FRDA and the pleiotropic effects of leriglitazone that could be a promising therapy for FRDA.

Open-label pilot study of oral methylprednisolone for the treatment of patients with friedreich ataxia.

INTRODUCTION: In this study we assessed the effect of methylprednisolone on safety, tolerability, and ability in Friedreich ataxia (FRDA). METHODS: The study was an open-label trial of pulse methylprednisolone on 11 participants with FRDA. All participants followed a 28-day treatment cycle, repeated 7 times. Patients were assessed with the timed 25-foot walk (T25FW), 1-minute walk (1MW), the Friedreich Ataxia Rating Scale (FARS), and the 9-hole peg test (9HPT). Efficacy was tested by comparing baseline and week 26 visits, separated into adult and pediatric groups. RESULTS: In comparisons of participants' baseline and week 26 visits, only the pediatric cohort's 1MW score showed change (P < 0.05). The T25FW, the primary outcome measure, did not change significantly. DISCUSSION: Pediatric participants improved their gait distance in the 1MW, but did not significantly improve in other measures in this overall negative study. Methylprednisolone was generally well tolerated, suggesting that it may be useful for ambulatory children with FRDA if benefit is found with further study.

Randomized, double-blind, placebo-controlled study of interferon-γ 1b in Friedreich Ataxia.

Objective: In vitro, in vivo, and open-label studies suggest that interferon gamma (IFN-γ 1b) may improve clinical features in Friedreich Ataxia through an increase in frataxin levels. The present study evaluates the efficacy and safety of IFN-γ 1b in the treatment of Friedreich Ataxia through a double-blind, multicenter, placebo-controlled trial. Methods: Ninety-two subjects with FRDA between 10 and 25 years of age were enrolled. Subjects received either IFN-γ 1b or placebo for 6 months. The primary outcome measure was the modified Friedreich Ataxia Rating Scale (mFARS). Results: No difference was noted between the groups after 6 months of treatment in the mFARS or secondary outcome measures. No change was noted in buccal cell or whole blood frataxin levels. However, during an open-label extension period, subjects had a more stable course than expected based on natural history data. Conclusions: This study provides no direct evidence for a beneficial effect of IFN-γ1b in FRDA. The modest stabilization compared to natural history data leaves open the possibility that longer studies may demonstrate benefit.

GRP75 overexpression rescues frataxin deficiency and mitochondrial phenotypes in Friedreich ataxia cellular models.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by the deficiency of frataxin, a mitochondrial protein crucial for iron-sulfur cluster biogenesis and adenosine triphosphate (ATP) production. Currently, there is no therapy to slow down the progression of FRDA. Recent evidence indicates that posttranslational regulation of residual frataxin levels can rescue some of the functional deficit of FRDA, raising the possibility of enhancing levels of residual frataxin as a treatment for FRDA. Here, we present evidence that mitochondrial molecular chaperone GRP75, also known as mortalin/mthsp70/PBP74, directly interacts with frataxin both in vivo in mouse cortex and in vitro in cortical neurons. Overexpressing GRP75 increases the levels of both wild-type frataxin and clinically relevant missense frataxin variants in human embryonic kidney 293 cells, while clinical GRP75 variants such as R126W, A476T and P509S impair the binding of GRP75 with frataxin and the effect of GRP75 on frataxin levels. In addition, GRP75 overexpression rescues frataxin deficiency and abnormal cellular phenotypes such as the abnormal mitochondrial network and decreased ATP levels in FRDA patient-derived cells. The effect of GRP75 on frataxin might be in part mediated by the physical interaction between GRP75 and mitochondrial processing peptidase (MPP), which makes frataxin more accessible to MPP. As GRP75 levels are decreased in multiple cell types of FRDA patients, restoring GRP75 might be effective in treating both typical FRDA patients with two guanine-adenine-adenine repeat expansions and compound heterozygous patients with point mutations.

Anti-NMDA Receptor Encephalitis: Clinical Features and Basic Mechanisms.

In slightly more than 10 years, anti-NMDA receptor (NMDAR) encephalitis has changed from a rare paraneoplastic syndrome to the most common cause of nonviral encephalitis. It presents fulminantly with progressive psychosis, seizures, and autonomic dysfunction, leading to death if untreated. However, rapid recognition and treatment can lead to survival and a return to baseline levels of functioning in many patients. While initially associated with ovarian teratomas, it is now associated with other tumors and can reflect a postviral event. The antibodies to the NMDAR made in this syndrome are pathogenic and are directed at the extracellular domain of the GluN1 subunit. Such antibodies lead to internalization of NMDARs in model systems, leading to a physiological state characterized by NMDAR hypofunction. Analogous disorders, characterized by antibodies to other synaptic receptors, present with neurological and psychiatric dysfunction and also appear to reflect antibody-induced internalization of receptors. However, this simple pathophysiology may be too simplistic to reflect the complexity of events in anti-NMDAR encephalitis. Future scientific investigations may allow a more complete understanding of this disorder and improve treatment of anti-NMDAR encephalitis.

Role of frataxin protein deficiency and metabolic dysfunction in Friedreich ataxia, an autosomal recessive mitochondrial disease.

Friedreich ataxia (FRDA) is a progressive neurodegenerative disease with developmental features caused by a genetic deficiency of frataxin, a small, nuclear-encoded mitochondrial protein. Frataxin deficiency leads to impairment of iron-sulphur cluster synthesis, and consequently, ATP production abnormalities. Based on the involvement of such processes in FRDA, initial pathophysiological hypotheses focused on reactive oxygen species (ROS) production as a key component of the mechanism. With further study, a variety of other events appear to be involved, including abnormalities of mitochondrially related metabolism and dysfunction in mitochondrial biogenesis. Consequently, present therapies focus not only on free radical damage, but also on control of metabolic abnormalities and correction of mitochondrial biogenesis. Understanding the multitude of abnormalities in FRDA thus offers possibilities for treatment of this disorder.

Early cerebellar deficits in mitochondrial biogenesis and respiratory chain complexes in the KIKO mouse model of Friedreich ataxia.

Friedreich ataxia (FRDA), the most common recessive inherited ataxia, results from deficiency of frataxin, a small mitochondrial protein crucial for iron-sulphur cluster formation and ATP production. Frataxin deficiency is associated with mitochondrial dysfunction in FRDA patients and animal models; however, early mitochondrial pathology in FRDA cerebellum remains elusive. Using frataxin knock-in/knockout (KIKO) mice and KIKO mice carrying the mitoDendra transgene, we show early cerebellar deficits in mitochondrial biogenesis and respiratory chain complexes in this FRDA model. At asymptomatic stages, the levels of PGC-1α (PPARGC1A), the mitochondrial biogenesis master regulator, are significantly decreased in cerebellar homogenates of KIKO mice compared with age-matched controls. Similarly, the levels of the PGC-1α downstream effectors, NRF1 and Tfam, are significantly decreased, suggesting early impaired cerebellar mitochondrial biogenesis pathways. Early mitochondrial deficiency is further supported by significant reduction of the mitochondrial markers GRP75 (HSPA9) and mitofusin-1 in the cerebellar cortex. Moreover, the numbers of Dendra-labeled mitochondria are significantly decreased in cerebellar cortex, confirming asymptomatic cerebellar mitochondrial biogenesis deficits. Functionally, complex I and II enzyme activities are significantly reduced in isolated mitochondria and tissue homogenates from asymptomatic KIKO cerebella. Structurally, levels of the complex I core subunit NUDFB8 and complex II subunits SDHA and SDHB are significantly lower than those in age-matched controls. These results demonstrate complex I and II deficiency in KIKO cerebellum, consistent with defects identified in FRDA patient tissues. Thus, our findings identify early cerebellar mitochondrial biogenesis deficits as a potential mediator of cerebellar dysfunction and ataxia, thereby providing a potential therapeutic target for early intervention of FRDA.

An evolutionary switch in ND2 enables Src kinase regulation of NMDA receptors.

The non-receptor tyrosine kinase Src is a key signalling hub for upregulating the function of N-methyl D-aspartate receptors (NMDARs). Src is anchored within the NMDAR complex via NADH dehydrogenase subunit 2 (ND2), a mitochondrially encoded adaptor protein. The interacting regions between Src and ND2 have been broadly identified, but the interaction between ND2 and the NMDAR has remained elusive. Here we generate a homology model of ND2 and dock it onto the NMDAR via the transmembrane domain of GluN1. This interaction is enabled by the evolutionary loss of three helices in bilaterian ND2 proteins compared to their ancestral homologues. We experimentally validate our model and demonstrate that blocking this interaction with an ND2 fragment identified in our experimental studies prevents Src-mediated upregulation of NMDAR currents in neurons. Our findings establish the mode of interaction between an NMDAR accessory protein with one of the core subunits of the receptor.

Treatment of inflammatory and neuropathic pain by uncoupling Src from the NMDA receptor complex.

Chronic pain hypersensitivity depends on N-methyl-D-aspartate receptors (NMDARs). However, clinical use of NMDAR blockers is limited by side effects resulting from suppression of the physiological functions of these receptors. Here we report a means to suppress pain hypersensitivity without blocking NMDARs, but rather by inhibiting the binding of a key enhancer of NMDAR function, the protein tyrosine kinase Src. We show that a peptide consisting of amino acids 40-49 of Src fused to the protein transduction domain of the HIV Tat protein (Src40-49Tat) prevented pain behaviors induced by intraplantar formalin and reversed pain hypersensitivity produced by intraplantar injection of complete Freund's adjuvant or by peripheral nerve injury. Src40-49Tat had no effect on basal sensory thresholds, acute nociceptive responses or cardiovascular, respiratory, locomotor or cognitive functions. Thus, through targeting of Src-mediated enhancement of NMDARs, inflammatory and neuropathic pain are suppressed without the deleterious consequences of directly blocking NMDARs, an approach that may be of broad relevance to managing chronic pain.

Developmental and cell-selective variations in N-methyl-D-aspartate receptor degradation by calpain.

NMDA receptors play critical roles in synaptic modulation and neurological disorders. In this study, we investigated the developmental changes in NR2 cleavage by NMDA receptor-activated calpain in cultured cortical and hippocampal neurons. Calpain activity increased with development, associated with increased expression of NMDA receptors but not of calpain I. The activation of calpain in immature and mature cortical cultures was inhibited by antagonists of NR1/2B and NR1/2A/2B receptors, whereas the inhibition of NR1/2B receptors did not alter calpain activation in mature hippocampal cultures. The degradation of NR2 subunits by calpain differed with developmental age. NR2A was not a substrate of calpain in mature hippocampal cultures, but was cleaved in immature cortical and hippocampal cultures. NR2B degradation by calpain in cortical cultures decreased with development, but the level of degradation of NR2B in hippocampal cultures did not change. The kinetics of NMDA receptor-gated whole cell currents were also modulated by calpain activation in a manner that varied with developmental stage in vitro. In early (but not later) developmental stages, calpain activation altered the NMDA-evoked current rise time and time constants for both desensitization and deactivation. Our data suggest that the susceptibility of the NMDA receptor to cleavage by calpain varies with neuronal maturity in a manner that may alter its electrophysiological properties.

Interactions of postsynaptic density-95 and the NMDA receptor 2 subunit control calpain-mediated cleavage of the NMDA receptor.

The calcium-dependent protease calpain cleaves the NMDA receptor 2 (NR2) subunit of the NMDA receptor both in vitro and in vivo and thus potentially modulates NMDA receptor function and turnover. We examined the ability of postsynaptic density-95 (PSD-95) protein to alter the calpain-mediated cleavage of NR2A and NR2B. Coexpression of PSD-95 with NMDA receptors in human embryonic kidney 293 cells blocked cleavage of NR2A and NR2B by NMDA receptor-activated calpain. NR2A cleavage by calpain occurred in the cell surface and intracellular fractions and required the presence of NR1 subunits. The blocking effect of PSD-95 did not result from decreased calpain activity, lowered intracellular calcium responses, or the blockade of internalization. Instead, this effect was eliminated by deletion of the C-terminal ESDV motif of NR2A or by overexpression of a palmitoylation-deficient PSD-95 mutant lacking the ability to cluster and to interact with NMDA receptors in situ, suggesting a role for association between the C terminus of NR2A and clustered PSD-95. Synapse-associated protein 102, a membrane-associated guanylate kinase interacting with NR2A but lacking palmitoylation motifs and the ability to cluster, did not protect NR2A from cleavage by calpain. Pharmacological inhibition of palmitoylation disrupted the interaction of PSD-95 with NMDA receptors in cortical neurons and allowed NR2A to be cleaved by calpain, whereas NR2A could not be cleaved in untreated neurons. These results indicate that PSD-95 clustering and direct association of NR2A and PSD-95 mediate the blocking effect of PSD-95 on calpain cleavage. PSD-95 could regulate the susceptibility of NMDA receptors to calpain-mediated cleavage during synaptic transmission and excitotoxicity.