Identification of Kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons.

Proteins usually form complexes to fulfill variable physiological functions. In neurons, communication relies on synapses where receptors, channels, and anchoring proteins form complexes to precisely control signal transduction, synaptic integration, and action potential firing. Although there are many published protocols to isolate protein complexes in cell lines, isolation in neurons has not been well established. Here we introduce a method that combines lentiviral protein expression with tandem affinity purification followed by mass-spectrometry (TAP-MS) to identify protein complexes in neurons. This protocol can also be used to identify post-translational modifications (PTMs) of synaptic proteins. We used the A-type voltage-gated K+ channel subunit Kv4.2 as the target protein. Kv4.2 is highly expressed in the hippocampus where it contributes to learning and memory through its regulation of neuronal excitability and synaptic plasticity. We tagged Kv4.2 with the calmodulin-binding-peptide (CBP) and streptavidin-binding-peptide (SBP) at its C-terminus and expressed it in neurons via lentivirus. Kv4.2 was purified by two-step TAP and samples were analyzed by MS. MS identified two prominently known Kv4.2 interacting proteins [dipeptidyl peptidase like (DPPs) and Kv channel-interacting proteins (KChIPs)] in addition to novel synaptic proteins including glutamate receptors, a calcium channel, and anchoring proteins. Co-immunoprecipitation and colocalization experiments validated the association of Kv4.2 with glutamate receptors. In addition to protein complex identification, we used TAP-MS to identify Kv4.2 phosphorylation sites. Several known and unknown phosphorylation sites were identified. These findings provide a novel path to identify protein-protein interactions and PTMs in neurons and shed light on mechanisms of neuronal signaling potentially involved in the pathology of neurological diseases.

Transport between im/mobile fractions shapes the speed and profile of cargo distribution in neurons.

Neuronal function requires continuous distribution of ion channels and other proteins throughout large cell morphologies. Protein distribution is complicated by immobilization of freely diffusing subunits such as on lipid rafts or in postsynaptic densities. Here, we infer rates of immobilization for the voltage-gated potassium channel Kv4.2. Fluorescence recovery after photobleaching quantifies protein diffusion kinetics, typically reported as a recovery rate and mobile fraction. We show that, implicit in the fluorescence recovery, are rates of particle transfer between mobile and immobile fractions (im/mobilization). We performed photobleaching of fluorescein-tagged ion channel Kv4.2-sGFP2 in over 450 dendrites of rat hippocampal cells. Using mass-action models, we infer rates of Kv4.2-sGFP2 im/mobilization. Using a realistic neuron morphology, we show how these rates shape the speed and profile of subunit distribution. The experimental protocol and model inference introduced here is widely applicable to other cargo and experimental systems.

Alzheimer's disease/dementia-associated brain pathology in aging DPP6-KO mice.

We have previously reported that the single transmembrane protein Dipeptidyl Peptidase Like 6 (DPP6) impacts neuronal and synaptic development. DPP6-KO mice are impaired in hippocampal-dependent learning and memory and exhibit smaller brain size. Recently, we have described novel structures in hippocampal area CA1 in aging mice, apparently derived from degenerating presynaptic terminals, that are significantly more prevalent in DPP6-KO mice compared to WT mice of the same age and that these structures were observed earlier in development in DPP6-KO mice. These novel structures appear as clusters of large puncta that colocalize NeuN, synaptophysin, and chromogranin A, and also partially label for MAP2, amyloid ß, APP, α-synuclein, and phosphorylated tau, with synapsin-1 and VGluT1 labeling on their periphery. In this current study, using immunofluorescence and electron microscopy, we confirm that both APP and amyloid ß are prevalent in these structures; and we show with immunofluorescence the presence of similar structures in humans with Alzheimer's disease. Here we also found evidence that aging DPP6-KO mutants show additional changes related to Alzheimer's disease. We used in vivo MRI to show reduced size of the DPP6-KO brain and hippocampus. Aging DPP6-KO hippocampi contained fewer total neurons and greater neuron death and had diagnostic biomarkers of Alzheimer's disease present including accumulation of amyloid ß and APP and increase in expression of hyper-phosphorylated tau. The amyloid ß and phosphorylated tau pathologies were associated with neuroinflammation characterized by increases in microglia and astrocytes. And levels of proinflammatory or anti-inflammatory cytokines increased in aging DPP6-KO mice. We finally show that aging DPP6-KO mice display circadian dysfunction, a common symptom of Alzheimer's disease. Together these results indicate that aging DPP6-KO mice show symptoms of enhanced neurodegeneration reminiscent of dementia associated with a novel structure resulting from synapse loss and neuronal death. This study continues our laboratory's work in discerning the function of DPP6 and here provides compelling evidence of a direct role of DPP6 in Alzheimer's disease.

Neuronal Roles of the Multifunctional Protein Dipeptidyl Peptidase-like 6 (DPP6).

The concerted action of voltage-gated ion channels in the brain is fundamental in controlling neuronal physiology and circuit function. Ion channels often associate in multi-protein complexes together with auxiliary subunits, which can strongly influence channel expression and function and, therefore, neuronal computation. One such auxiliary subunit that displays prominent expression in multiple brain regions is the Dipeptidyl aminopeptidase-like protein 6 (DPP6). This protein associates with A-type K+ channels to control their cellular distribution and gating properties. Intriguingly, DPP6 has been found to be multifunctional with an additional, independent role in synapse formation and maintenance. Here, we feature the role of DPP6 in regulating neuronal function in the context of its modulation of A-type K+ channels as well as its independent involvement in synaptic development. The prevalence of DPP6 in these processes underscores its importance in brain function, and recent work has identified that its dysfunction is associated with host of neurological disorders. We provide a brief overview of these and discuss research directions currently underway to advance our understanding of the contribution of DPP6 to their etiology.

R-type voltage-gated Ca2+ channels mediate A-type K+ current regulation of synaptic input in hippocampal dendrites.

The subthreshold voltage-gated transient K+ current (IA) carried by pore-forming Kv4.2 subunits regulates the propagation of synaptic input, dendritic excitability, and synaptic plasticity in CA1 pyramidal neuron dendrites of the hippocampus. We report that the Ca2+ channel subunit Cav2.3 regulates IA in this cell type. We initially identified Cav2.3 as a Kv4.2-interacting protein in a proteomic screen and we confirmed Cav2.3-Kv4.2 complex association using multiple techniques. Functionally, Cav2.3 Ca2+-entry increases Kv4.2-mediated whole-cell current due to an increase in Kv4.2 surface expression. Using pharmacology and Cav2.3 knockout mice, we show that Cav2.3 regulates the dendritic gradient of IA. Furthermore, the loss of Cav2.3 function leads to the enhancement of AMPA receptor-mediated synaptic currents and NMDA receptor-mediated spine Ca2+ influx. These results propose that Cav2.3 and Kv4.2 are integral constituents of an ion channel complex that affects synaptic function in the hippocampus.

Cushing Syndrome in a Pediatric Patient With a KCNJ5 Variant and Successful Treatment With Low-dose Ketoconazole.

CONTEXT: Pathogenic variants in KCNJ5, encoding the GIRK4 (Kir3.4) potassium channel, have been implicated in the pathogenesis of familial hyperaldosteronism type-III (FH-III) and sporadic primary aldosteronism (PA). In addition to aldosterone, glucocorticoids are often found elevated in PA in association with KCNJ5 pathogenic variants, albeit at subclinical levels. However, to date no GIRK4 defects have been linked to Cushing syndrome (CS). PATIENT: We present the case of a 10-year-old child who presented with CS at an early age due to bilateral adrenocortical hyperplasia (BAH). The patient was placed on low-dose ketoconazole (KZL), which controlled hypercortisolemia and CS-related signs. Discontinuation of KZL for even 6 weeks led to recurrent CS. RESULTS: Screening for known genes causing cortisol-producing BAHs (PRKAR1A, PRKACA, PRKACB, PDE11A, PDE8B, ARMC5) failed to identify any gene defects. Whole-exome sequencing showed a novel KCNJ5 pathogenic variant (c.506T>C, p.L169S) inherited from her father. In vitro studies showed that the p.L169S variant affects conductance of the Kir3.4 channel without affecting its expression or membrane localization. Although there were no effects on steroidogenesis in vitro, there were modest changes in protein kinase A activity. In silico analysis of the mutant channel proposed mechanisms for the altered conductance. CONCLUSION: We present a pediatric patient with CS due to BAH and a germline defect in KCNJ5. Molecular investigations of this KCNJ5 variant failed to show a definite cause of her CS. However, this KCNJ5 variant differed in its function from KCNJ5 defects leading to PA. We speculate that GIRK4 (Kir3.4) may play a role in early human adrenocortical development and zonation and participate in the pathogenesis of pediatric BAH.

A novel structure associated with aging is augmented in the DPP6-KO mouse brain.

In addition to its role as an auxiliary subunit of A-type voltage-gated K+ channels, we have previously reported that the single transmembrane protein Dipeptidyl Peptidase Like 6 (DPP6) impacts neuronal and synaptic development. DPP6-KO mice are impaired in hippocampal-dependent learning and memory and exhibit smaller brain size. Using immunofluorescence and electron microscopy, we report here a novel structure in hippocampal area CA1 that was significantly more prevalent in aging DPP6-KO mice compared to WT mice of the same age and that these structures were observed earlier in development in DPP6-KO mice. These novel structures appeared as clusters of large puncta that colocalized NeuN, synaptophysin, and chromogranin A. They also partially labeled for MAP2, and with synapsin-1 and VGluT1 labeling on their periphery. Electron microscopy revealed that these structures are abnormal, enlarged presynaptic swellings filled with mainly fibrous material with occasional peripheral, presynaptic active zones forming synapses. Immunofluorescence imaging then showed that a number of markers for aging and especially Alzheimer's disease were found as higher levels in these novel structures in aging DPP6-KO mice compared to WT. Together these results indicate that aging DPP6-KO mice have increased numbers of novel, abnormal presynaptic structures associated with several markers of Alzheimer's disease.

P38 Regulates Kainic Acid-Induced Seizure and Neuronal Firing via Kv4.2 Phosphorylation.

The subthreshold, transient A-type K+ current is a vital regulator of the excitability of neurons throughout the brain. In mammalian hippocampal pyramidal neurons, this current is carried primarily by ion channels comprising Kv4.2 α-subunits. These channels occupy the somatodendritic domains of these principle excitatory neurons and thus regulate membrane voltage relevant to the input-output efficacy of these cells. Owing to their robust control of membrane excitability and ubiquitous expression in the hippocampus, their dysfunction can alter network stability in a manner that manifests in recurrent seizures. Indeed, growing evidence implicates these channels in intractable epilepsies of the temporal lobe, which underscores the importance of determining the molecular mechanisms underlying their regulation and contribution to pathologies. Here, we describe the role of p38 kinase phosphorylation of a C-terminal motif in Kv4.2 in modulating hippocampal neuronal excitability and behavioral seizure strength. Using a combination of biochemical, single-cell electrophysiology, and in vivo seizure techniques, we show that kainic acid-induced seizure induces p38-mediated phosphorylation of Thr607 in Kv4.2 in a time-dependent manner. The pharmacological and genetic disruption of this process reduces neuronal excitability and dampens seizure intensity, illuminating a cellular cascade that may be targeted for therapeutic intervention to mitigate seizure intensity and progression.

Activity-dependent isomerization of Kv4.2 by Pin1 regulates cognitive flexibility.

Voltage-gated K+ channels function in macromolecular complexes with accessory subunits to regulate brain function. Here, we describe a peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1)-dependent mechanism that regulates the association of the A-type K+ channel subunit Kv4.2 with its auxiliary subunit dipeptidyl peptidase 6 (DPP6), and thereby modulates neuronal excitability and cognitive flexibility. We show that activity-induced Kv4.2 phosphorylation triggers Pin1 binding to, and isomerization of, Kv4.2 at the pThr607-Pro motif, leading to the dissociation of the Kv4.2-DPP6 complex. We generated a novel mouse line harboring a knock-in Thr607 to Ala (Kv4.2TA) mutation that abolished dynamic Pin1 binding to Kv4.2. CA1 pyramidal neurons of the hippocampus from these mice exhibited altered Kv4.2-DPP6 interaction, increased A-type K+ current, and reduced neuronal excitability. Behaviorally, Kv4.2TA mice displayed normal initial learning but improved reversal learning in both Morris water maze and lever press paradigms. These findings reveal a Pin1-mediated mechanism regulating reversal learning and provide potential targets for the treatment of neuropsychiatric disorders characterized by cognitive inflexibility.

Disruption of GpI mGluR-Dependent Cav2.3 Translation in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is an inherited intellectual impairment that results from the loss of fragile X mental retardation protein (FMRP), an mRNA binding protein that regulates mRNA translation at synapses. The absence of FMRP leads to neuronal and circuit-level hyperexcitability that is thought to arise from the aberrant expression and activity of voltage-gated ion channels, although the identification and characterization of these ion channels have been limited. Here, we show that FMRP binds the mRNA of the R-type voltage-gated calcium channel Cav2.3 in mouse brain synaptoneurosomes and represses Cav2.3 translation under basal conditions. Consequently, in hippocampal neurons from male and female FMRP KO mice, we find enhanced Cav2.3 protein expression by western blotting and abnormally large R currents in whole-cell voltage-clamp recordings. In agreement with previous studies showing that FMRP couples Group I metabotropic glutamate receptor (GpI mGluR) signaling to protein translation, we find that GpI mGluR stimulation results in increased Cav2.3 translation and R current in hippocampal neurons which is disrupted in FMRP KO mice. Thus, FMRP serves as a key translational regulator of Cav2.3 expression under basal conditions and in response to GpI mGluR stimulation. Loss of regulated Cav2.3 expression could underlie the neuronal hyperactivity and aberrant calcium spiking in FMRP KO mice and contribute to FXS, potentially serving as a novel target for future therapeutic strategies.SIGNIFICANCE STATEMENT Patients with fragile X syndrome (FXS) exhibit signs of neuronal and circuit hyperexcitability, including anxiety and hyperactive behavior, attention deficit disorder, and seizures. FXS is caused by the loss of fragile X mental retardation protein (FMRP), an mRNA binding protein, and the neuronal hyperexcitability observed in the absence of FMRP likely results from its ability to regulate the expression and activity of voltage-gated ion channels. Here we find that FMRP serves as a key translational regulator of the voltage-gated calcium channel Cav2.3 under basal conditions and following activity. Cav2.3 impacts cellular excitability and calcium signaling, and the alterations in channel translation and expression observed in the absence of FMRP could contribute to the neuronal hyperactivity that underlies FXS.

A novel bungarotoxin binding site-tagged construct reveals MAPK-dependent Kv4.2 trafficking.

Kv4.2 voltage-gated K+ channel subunits, the primary source of the somatodendritic A-type K+ current in CA1 pyramidal neurons of the hippocampus, play important roles in regulating dendritic excitability and plasticity. To better study the trafficking and subcellular distribution of Kv4.2, we created and characterized a novel Kv4.2 construct encoding a bungarotoxin binding site in the extracellular S3-S4 linker region of the α-subunit. When expressed, this construct can be visualized in living cells after staining with rhodamine-conjugated bungarotoxin. We validated the utility of this construct by visualizing the spontaneous internalization and insertion of Kv4.2 in HEK 293T cells. We further report that Kv4.2 colocalized with several endosome markers in HEK 293T cells. In addition, Kv4.2 internalization is significantly impaired by mitogen-activated protein kinase (MAPK) inhibitors in transfected primary hippocampal neurons. Therefore, this newly developed BBS-Kv4.2 construct provides a novel and powerful tool for studying surface Kv4.2 channel localization and trafficking.

Functional Coupling of Cav2.3 and BK Potassium Channels Regulates Action Potential Repolarization and Short-Term Plasticity in the Mouse Hippocampus.

Voltage-gated ion channels are essential for signal generation and propagation in neurons and other excitable cells. The high-voltage activated calcium-channel Cav2.3 is expressed throughout the central and peripheral nervous system, and within CA1 hippocampal pyramidal neurons it is localized throughout the somato-dendritic region and dendritic spines. Cav2.3 has been shown to provide calcium for other calcium-dependent potassium channels including small-conductance calcium-activated potassium channels (SK), but big-conductance calcium-activated potassium channels (BK) have been thought to be activated by calcium from all known voltage-gated calcium channels, except Cav2.3. Here we show for the first time that CA1 pyramidal cells which lack Cav2.3 show altered action potential (AP) waveforms, which can be traced back to reduced SK- and BK-channel function. This change in AP waveform leads to strengthened synaptic transmission between CA1 and the subiculum, resulting in increased short-term plasticity. Our results demonstrate that Cav2.3 impacts cellular excitability through functional interaction with BK channels, impacting communication between hippocampal subregions.

A polybasic motif in alternatively spliced KChIP2 isoforms prevents Ca2+ regulation of Kv4 channels.

The Kv4 family of A-type voltage-gated K+ channels regulates the excitability in hippocampal pyramidal neuron dendrites and are key determinants of dendritic integration, spike timing-dependent plasticity, long-term potentiation, and learning. Kv4.2 channel expression is down-regulated following hippocampal seizures and in epilepsy, suggesting A-type currents as therapeutic targets. In addition to pore-forming Kv4 subunits, modulatory auxiliary subunits called K+ channel-interacting proteins (KChIPs) modulate Kv4 expression and activity and are required to recapitulate native hippocampal A-type currents in heterologous expression systems. KChIP mRNAs contain multiple start sites and alternative exons that generate considerable N-terminal variation and functional diversity in shaping Kv4 currents. As members of the EF-hand domain-containing neuronal Ca2+ sensor protein family, KChIP auxiliary proteins may convey Ca2+ sensitivity upon Kv4 channels; however, to what degree intracellular Ca2+ regulates KChIP-Kv4.2 complexes is unclear. To answer this question, we expressed KChIP2 with Kv4.2 in HEK293T cells, and, with whole-cell patch-clamp electrophysiology, measured an ∼1.5-fold increase in Kv4.2 current density in the presence of elevated intracellular Ca2+ Intriguingly, the Ca2+ regulation of Kv4 current was specific to KChIP2b and KChIP2c splice isoforms that lack a putative polybasic domain that is present in longer KChIP2a1 and KChIP2a isoforms. Site-directed acidification of the basic residues within the polybasic motif of KChIP2a1 rescued Ca2+-mediated regulation of Kv4 current density. These results support divergent Ca2+ regulation of Kv4 channels mediated by alternative splicing of KChIP2 isoforms. They suggest that distinct KChIP-Kv4 interactions may differentially control excitability and function of hippocampal dendrites.

DPP6 Loss Impacts Hippocampal Synaptic Development and Induces Behavioral Impairments in Recognition, Learning and Memory.

DPP6 is well known as an auxiliary subunit of Kv4-containing, A-type K+ channels which regulate dendritic excitability in hippocampal CA1 pyramidal neurons. We have recently reported, however, a novel role for DPP6 in regulating dendritic filopodia formation and stability, affecting synaptic development and function. These results are notable considering recent clinical findings associating DPP6 with neurodevelopmental and intellectual disorders. Here we assessed the behavioral consequences of DPP6 loss. We found that DPP6 knockout (DPP6-KO) mice are impaired in hippocampus-dependent learning and memory. Results from the Morris water maze and T-maze tasks showed that DPP6-KO mice exhibit slower learning and reduced memory performance. DPP6 mouse brain weight is reduced throughout development compared with WT, and in vitro imaging results indicated that DPP6 loss affects synaptic structure and motility. Taken together, these results show impaired synaptic development along with spatial learning and memory deficiencies in DPP6-KO mice.

FRMPD4 mutations cause X-linked intellectual disability and disrupt dendritic spine morphogenesis.

FRMPD4 (FERM and PDZ Domain Containing 4) is a neural scaffolding protein that interacts with PSD-95 to positively regulate dendritic spine morphogenesis, and with mGluR1/5 and Homer to regulate mGluR1/5 signaling. We report the genetic and functional characterization of 4 FRMPD4 deleterious mutations that cause a new X-linked intellectual disability (ID) syndrome. These mutations were found to be associated with ID in ten affected male patients from four unrelated families, following an apparent X-linked mode of inheritance. Mutations include deletion of an entire coding exon, a nonsense mutation, a frame-shift mutation resulting in premature termination of translation, and a missense mutation involving a highly conserved amino acid residue neighboring FRMPD4-FERM domain. Clinical features of these patients consisted of moderate to severe ID, language delay and seizures alongside with behavioral and/or psychiatric disturbances. In-depth functional studies showed that a frame-shift mutation, FRMPD4p.Cys618ValfsX8, results in a disruption of FRMPD4 binding with PSD-95 and HOMER1, and a failure to increase spine density in transfected hippocampal neurons. Behavioral studies of frmpd4-KO mice identified hippocampus-dependent spatial learning and memory deficits in Morris Water Maze test. These findings point to an important role of FRMPD4 in normal cognitive development and function in humans and mice, and support the hypothesis that FRMPD4 mutations cause ID by disrupting dendritic spine morphogenesis in glutamatergic neurons.

Idiopathic Autism: Cellular and Molecular Phenotypes in Pluripotent Stem Cell-Derived Neurons.

Autism spectrum disorder is a complex neurodevelopmental disorder whose pathophysiology remains elusive as a consequence of the unavailability for study of patient brain neurons; this deficit may potentially be circumvented by neural differentiation of induced pluripotent stem cells. Rare syndromes with single gene mutations and autistic symptoms have significantly advanced the molecular and cellular understanding of autism spectrum disorders; however, in aggregate, they only represent a fraction of all cases of autism. In an effort to define the cellular and molecular phenotypes in human neurons of non-syndromic autism, we generated induced pluripotent stem cells (iPSCs) from three male autism spectrum disorder patients who had no identifiable clinical syndromes, and their unaffected male siblings and subsequently differentiated these patient-specific stem cells into electrophysiologically active neurons. iPSC-derived neurons from these autistic patients displayed decreases in the frequency and kinetics of spontaneous excitatory postsynaptic currents relative to controls, as well as significant decreases in Na+ and inactivating K+ voltage-gated currents. Moreover, whole-genome microarray analysis of gene expression identified 161 unique genes that were significantly differentially expressed in autistic patient iPSC-derived neurons (>twofold, FDR < 0.05). These genes were significantly enriched for processes related to synaptic transmission, such as neuroactive ligand-receptor signaling and extracellular matrix interactions, and were enriched for genes previously associated with autism spectrum disorder. Our data demonstrate aberrant voltage-gated currents and underlying molecular changes related to synaptic function in iPSC-derived neurons from individuals with idiopathic autism as compared to unaffected siblings controls.

Functional characterization of two novel germline mutations of the KCNJ5 gene in hypertensive patients without primary aldosteronism but with ACTH-dependent aldosterone hypersecretion.

BACKGROUND: Germline mutations of the KCNJ5 gene encoding Kir3·4, a member of the inwardly rectifying K+ channel, have been identified in 'normal' adrenal glands, patients with familial hyperaldosteronism (FH) type III, aldosterone-producing adenomas (APAs) and sporadic cases of primary aldosteronism (PA). OBJECTIVE: To present two novel KCNJ5 gene mutations in hypertensive patients without PA, but with Adrenocorticotropic hormone (ACTH)-dependent aldosterone hypersecretion. DESIGN AND PATIENTS: Two hypertensive patients without PA, who exhibited enhanced ACTH-dependent response of aldosterone secretion, underwent genetic testing for the presence of the CYP11B1/CYP11B2 chimeric gene and KCNJ5 gene mutations. Genomic DNA was isolated from peripheral white blood cells, and the exons of the entire coding regions of the above genes were amplified and sequenced. Electrophysiological studies were performed to determine the effect of identified mutation(s) on the membrane reversal potentials. Structural biology studies were also carried out. RESULTS: Two novel germline heterozygous KCNJ5 mutations, p.V259M and p.Y348N, were detected in the two subjects. Electrophysiological studies showed that the Y348N mutation resulted in significantly less negative reversal potentials, suggesting loss of ion selectivity, while the V259M mutation did not affect the Kir3.4 current. In the mutated structural biology model, the N348 mutant resulted in significant loss of the ability for hydrogen bonding, while the M259 mutant was capable of establishing weaker interactions. The CYP11B1/CYP11B2 chimeric gene was not detected. CONCLUSIONS: These findings expand on the clinical spectrum of phenotypes associated with KCNJ5 mutations and implicate these mutations in the pathogenesis of hypertension associated with increased aldosterone response to ACTH stimulation.

Matrix Metalloproteinase-9 Regulates Neuronal Circuit Development and Excitability.

In early postnatal development, naturally occurring cell death, dendritic outgrowth, and synaptogenesis sculpt neuronal ensembles into functional neuronal circuits. Here, we demonstrate that deletion of the extracellular proteinase matrix metalloproteinase-9 (MMP-9) affects each of these processes, resulting in maladapted neuronal circuitry. MMP-9 deletion increases the number of CA1 pyramidal neurons but decreases dendritic length and complexity. Parallel changes in neuronal morphology are observed in primary visual cortex and persist into adulthood. Individual CA1 neurons in MMP-9(-/-) mice have enhanced input resistance and a significant increase in the frequency, but not amplitude, of miniature excitatory postsynaptic currents (mEPSCs). Additionally, deletion of MMP-9 significantly increases spontaneous neuronal activity in awake MMP-9(-/-) mice and enhances response to acute challenge by the excitotoxin kainate. Our data document a novel role for MMP-9-dependent proteolysis: the regulation of several aspects of circuit maturation to constrain excitability throughout life.

Human endogenous retrovirus-K contributes to motor neuron disease.

The role of human endogenous retroviruses (HERVs) in disease pathogenesis is unclear. We show that HERV-K is activated in a subpopulation of patients with sporadic amyotrophic lateral sclerosis (ALS) and that its envelope (env) protein may contribute to neurodegeneration. The virus was expressed in cortical and spinal neurons of ALS patients, but not in neurons from control healthy individuals. Expression of HERV-K or its env protein in human neurons caused retraction and beading of neurites. Transgenic animals expressing the env gene developed progressive motor dysfunction accompanied by selective loss of volume of the motor cortex, decreased synaptic activity in pyramidal neurons, dendritic spine abnormalities, nucleolar dysfunction, and DNA damage. Injury to anterior horn cells in the spinal cord was manifested by muscle atrophy and pathological changes consistent with nerve fiber denervation and reinnervation. Expression of HERV-K was regulated by TAR (trans-activation responsive) DNA binding protein 43, which binds to the long terminal repeat region of the virus. Thus, HERV-K expression within neurons of patients with ALS may contribute to neurodegeneration and disease pathogenesis.

Tau-dependent Kv4.2 depletion and dendritic hyperexcitability in a mouse model of Alzheimer's disease.

Neuronal hyperexcitability occurs early in the pathogenesis of Alzheimer's disease (AD) and contributes to network dysfunction in AD patients. In other disorders with neuronal hyperexcitability, dysfunction in the dendrites often contributes, but dendritic excitability has not been directly examined in AD models. We used dendritic patch-clamp recordings to measure dendritic excitability in the CA1 region of the hippocampus. We found that dendrites, more so than somata, of hippocampal neurons were hyperexcitable in mice overexpressing Aß. This dendritic hyperexcitability was associated with depletion of Kv4.2, a dendritic potassium channel important for regulating dendritic excitability and synaptic plasticity. The antiepileptic drug, levetiracetam, blocked Kv4.2 depletion. Tau was required, as crossing with tau knock-out mice also prevented both Kv4.2 depletion and dendritic hyperexcitability. Dendritic hyperexcitability induced by Kv4.2 deficiency exacerbated behavioral deficits and increased epileptiform activity in hAPP mice. We conclude that increased dendritic excitability, associated with changes in dendritic ion channels including Kv4.2, may contribute to neuronal dysfunction in early stages AD.