A modified mouse model of Friedreich's ataxia with conditional Fxn allele homozygosity delays onset of cardiomyopathy.

Friedreich's ataxia (FA) is an autosomal recessive disorder caused by a deficiency in frataxin (FXN), a mitochondrial protein that plays a critical role in the synthesis of iron-sulfur clusters (Fe-S), vital inorganic cofactors necessary for numerous cellular processes. FA is characterized by progressive ataxia and hypertrophic cardiomyopathy, with cardiac dysfunction as the most common cause of mortality in patients. Commonly used cardiac-specific mouse models of FA use the muscle creatine kinase (MCK) promoter to express Cre recombinase in cardiomyocytes and striated muscle cells in mice with one conditional Fxn allele and one floxed-out/null allele. These mice quickly develop cardiomyopathy that becomes fatal by 9-11 wk of age. Here, we generated a cardiac-specific model with floxed Fxn allele homozygosity (MCK-Fxnflox/flox). MCK-Fxnflox/flox mice were phenotypically normal at 9 wk of age, despite no detectable FXN protein expression. Between 13 and 15 wk of age, these mice began to display progressive cardiomyopathy, including decreased ejection fraction and fractional shortening and increased left ventricular mass. MCK-Fxnflox/flox mice began to lose weight around 16 wk of age, characteristically associated with heart failure in other cardiac-specific FA models. By 18 wk of age, MCK-Fxnflox/flox mice displayed elevated markers of Fe-S deficiency, cardiac stress and injury, and cardiac fibrosis. This modified model reproduced important pathophysiological and biochemical features of FA over a longer timescale than previous cardiac-specific mouse models, offering a larger window for studying potential therapeutics.NEW & NOTEWORTHY Previous cardiac-specific frataxin knockout models exhibit rapid and fatal cardiomyopathy by 9 wk of age. This severe phenotype poses challenges for the design and execution of intervention studies. We introduce an alternative cardiac-specific model, MCK-Fxnflox/flox, with increased longevity and delayed onset of all major phenotypes. These phenotypes develop to the same severity as previous models. Thus, this new model provides the same cardiomyopathy-associated mortality with a larger window for potential studies.

Clustering of CaV 1.3 L-type calcium channels by Shank3.

Clustering of L-type voltage-gated Ca2+ channels (LTCCs) in the plasma membrane is increasingly implicated in creating highly localized Ca2+ signaling nanodomains. For example, neuronal LTCC activation can increase phosphorylation of the nuclear CREB transcription factor by increasing Ca2+ concentrations within a nanodomain close to the channel, without requiring bulk Ca2+ increases in the cytosol or nucleus. However, the molecular basis for LTCC clustering is poorly understood. The postsynaptic scaffolding protein Shank3 specifically associates with one of the major neuronal LTCCs, the CaV 1.3 calcium channel, and is required for optimal LTCC-dependent excitation-transcription coupling. Here, we co-expressed CaV 1.3 α1 subunits with two distinct epitope-tags with or without Shank3 in HEK cells. Co-immunoprecipitation studies using the cell lysates revealed that Shank3 can assemble complexes containing multiple CaV 1.3 α1 subunits under basal conditions. Moreover, CaV 1.3 LTCC complex formation was facilitated by CaV ß subunits (ß3 and ß2a), which also interact with Shank3. Shank3 interactions with CaV 1.3 LTCCs and multimeric CaV 1.3 LTCC complex assembly were disrupted following the addition of Ca2+ to cell lysates, perhaps simulating conditions within an activated CaV 1.3 LTCC nanodomain. In intact HEK293T cells, co-expression of Shank3 enhanced the intensity of membrane-localized CaV 1.3 LTCC clusters under basal conditions, but not after Ca2+ channel activation. Live cell imaging studies also revealed that Ca2+ influx through LTCCs disassociated Shank3 from CaV 1.3 LTCCs clusters and reduced the CaV 1.3 cluster intensity. Deletion of the Shank3 PDZ domain prevented both binding to CaV 1.3 and the changes in multimeric CaV 1.3 LTCC complex assembly in vitro and in HEK293 cells. Finally, we found that shRNA knock-down of Shank3 expression in cultured rat primary hippocampal neurons reduced the intensity of surface-localized CaV 1.3 LTCC clusters in dendrites. Taken together, our findings reveal a novel molecular mechanism contributing to neuronal LTCC clustering under basal conditions.

In vivo overexpression of frataxin causes toxicity mediated by iron-sulfur cluster deficiency.

Friedreich's ataxia is a rare disorder resulting from deficiency of frataxin, a mitochondrial protein implicated in the synthesis of iron-sulfur clusters. Preclinical studies in mice have shown that gene therapy is a promising approach to treat individuals with Friedreich's ataxia. However, a recent report provided evidence that AAVrh10-mediated overexpression of frataxin could lead to cardiotoxicity associated with mitochondrial dysfunction. While evaluating an AAV9-based frataxin gene therapy using a chicken ß-actin promoter, we showed that toxic overexpression of frataxin could be reached in mouse liver and heart with doses between 1 × 1013 and 1 × 1014 vg/kg. In a mouse model of cardiac disease, these doses only corrected cardiac dysfunction partially and transiently and led to adverse findings associated with iron-sulfur cluster deficiency in liver. We demonstrated that toxicity required frataxin's primary function by using a frataxin construct bearing the N146K mutation, which impairs binding to the iron-sulfur cluster core complex. At the lowest tested dose, we observed moderate liver toxicity that was accompanied by progressive loss of transgene expression and liver regeneration. Together, our data provide insights into the toxicity of frataxin overexpression that should be considered in the development of a gene therapy approach for Friedreich's ataxia.

Neuronal L-Type Calcium Channel Signaling to the Nucleus Requires a Novel CaMKIIα-Shank3 Interaction.

The activation of neuronal plasma membrane Ca2+ channels stimulates many intracellular responses. Scaffolding proteins can preferentially couple specific Ca2+ channels to distinct downstream outputs, such as increased gene expression, but the molecular mechanisms that underlie the exquisite specificity of these signaling pathways are incompletely understood. Here, we show that complexes containing CaMKII and Shank3, a postsynaptic scaffolding protein known to interact with L-type calcium channels (LTCCs), can be specifically coimmunoprecipitated from mouse forebrain extracts. Activated purified CaMKIIα also directly binds Shank3 between residues 829 and 1130. Mutation of Shank3 residues 949Arg-Arg-Lys951 to three alanines disrupts CaMKII binding in vitro and CaMKII association with Shank3 in heterologous cells. Our shRNA/rescue studies revealed that Shank3 binding to both CaMKII and LTCCs is important for increased phosphorylation of the nuclear CREB transcription factor and expression of c-Fos induced by depolarization of cultured hippocampal neurons. Thus, this novel CaMKII-Shank3 interaction is essential for the initiation of a specific long-range signal from LTCCs in the plasma membrane to the nucleus that is required for activity-dependent changes in neuronal gene expression during learning and memory.SIGNIFICANCE STATEMENT Precise neuronal expression of genes is essential for normal brain function. Proteins involved in signaling pathways that underlie activity-dependent gene expression, such as CaMKII, Shank3, and L-type calcium channels, are often mutated in multiple neuropsychiatric disorders. Shank3 and CaMKII were previously shown to bind L-type calcium channels, and we show here that Shank3 also binds to CaMKII. Our data show that each of these interactions is required for depolarization-induced phosphorylation of the CREB nuclear transcription factor, which stimulates the expression of c-Fos, a neuronal immediate early gene with key roles in synaptic plasticity, brain development, and behavior.

CaMKIIα phosphorylation of Shank3 modulates ABI1-Shank3 interaction.

Protein-protein interactions can be modulated by phosphorylation of either binding partner, thereby altering subcellular localization and/or physiological function. Shank3, a master postsynaptic scaffolding protein that controls the developmental maturation of excitatory synapses, was recently shown to be phosphorylated by Protein Kinase A (PKA) at Ser685 in vivo. Mutation of Shank3 Ser685 was shown to modulate the binding of Abelson interactor 1 (ABI1), a component of the WAVE regulatory complex for actin remodeling, but a direct effect of Ser685 phosphorylation on ABI1 binding was not investigated. Here, we demonstrate that Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) also phosphorylates Shank3 at Ser685. Mutation of Ser685 to phospho-null alanine (S685A) prevented both CaMKIIα and PKA phosphorylation of a GST-Shank3 fusion protein. The co-immunoprecipitation of ABI1 with Shank3 from HEK293 cell extracts is reduced by mutation of Ser685 to either Ala or Asp. However, pre-phosphorylation of GST-Shank3 by purified CaMKIIα significantly increased binding of ABI1, and this effect was abrogated by Ser685 to Ala mutation in GST-Shank3. Taken together, our data suggest that neuronal ABI1-Shank3 interactions may be convergently regulated by Shank3 Ser685 phosphorylation in response to both Ca2+ and cAMP signaling, potentially modulating dendritic spine morphology.

A novel mechanism for Ca2+/calmodulin-dependent protein kinase II targeting to L-type Ca2+ channels that initiates long-range signaling to the nucleus.

Neuronal excitation can induce new mRNA transcription, a phenomenon called excitation-transcription (E-T) coupling. Among several pathways implicated in E-T coupling, activation of voltage-gated L-type Ca2+ channels (LTCCs) in the plasma membrane can initiate a signaling pathway that ultimately increases nuclear CREB phosphorylation and, in most cases, expression of immediate early genes. Initiation of this long-range pathway has been shown to require recruitment of Ca2+-sensitive enzymes to a nanodomain in the immediate vicinity of the LTCC by an unknown mechanism. Here, we show that activated Ca2+/calmodulin-dependent protein kinase II (CaMKII) strongly interacts with a novel binding motif in the N-terminal domain of CaV1 LTCC α1 subunits that is not conserved in CaV2 or CaV3 voltage-gated Ca2+ channel subunits. Mutations in the CaV1.3 α1 subunit N-terminal domain or in the CaMKII catalytic domain that largely prevent the in vitro interaction also disrupt CaMKII association with intact LTCC complexes isolated by immunoprecipitation. Furthermore, these same mutations interfere with E-T coupling in cultured hippocampal neurons. Taken together, our findings define a novel molecular interaction with the neuronal LTCC that is required for the initiation of a long-range signal to the nucleus that is critical for learning and memory.

A Novel Human CAMK2A Mutation Disrupts Dendritic Morphology and Synaptic Transmission, and Causes ASD-Related Behaviors.

Characterizing the functional impact of novel mutations linked to autism spectrum disorder (ASD) provides a deeper mechanistic understanding of the underlying pathophysiological mechanisms. Here we show that a de novo Glu183 to Val (E183V) mutation in the CaMKIIα catalytic domain, identified in a proband diagnosed with ASD, decreases both CaMKIIα substrate phosphorylation and regulatory autophosphorylation, and that the mutated kinase acts in a dominant-negative manner to reduce CaMKIIα-WT autophosphorylation. The E183V mutation also reduces CaMKIIα binding to established ASD-linked proteins, such as Shank3 and subunits of l-type calcium channels and NMDA receptors, and increases CaMKIIα turnover in intact cells. In cultured neurons, the E183V mutation reduces CaMKIIα targeting to dendritic spines. Moreover, neuronal expression of CaMKIIα-E183V increases dendritic arborization and decreases both dendritic spine density and excitatory synaptic transmission. Mice with a knock-in CaMKIIα-E183V mutation have lower total forebrain CaMKIIα levels, with reduced targeting to synaptic subcellular fractions. The CaMKIIα-E183V mice also display aberrant behavioral phenotypes, including hyperactivity, social interaction deficits, and increased repetitive behaviors. Together, these data suggest that CaMKIIα plays a previously unappreciated role in ASD-related synaptic and behavioral phenotypes.SIGNIFICANCE STATEMENT Many autism spectrum disorder (ASD)-linked mutations disrupt the function of synaptic proteins, but no single gene accounts for >1% of total ASD cases. The molecular networks and mechanisms that couple the primary deficits caused by these individual mutations to core behavioral symptoms of ASD remain poorly understood. Here, we provide the first characterization of a mutation in the gene encoding CaMKIIα linked to a specific neuropsychiatric disorder. Our findings demonstrate that this ASD-linked de novo CAMK2A mutation disrupts multiple CaMKII functions, induces synaptic deficits, and causes ASD-related behavioral alterations, providing novel insights into the synaptic mechanisms contributing to ASD.