Reducing filamin A restores cortical synaptic connectivity and early social communication following cellular mosaicism in ASD pathways.

Communication in the form of non-verbal, social vocalization, or crying is evolutionary conserved in mammals and is impaired early in human infants that are later diagnosed with autism spectrum disorder (ASD). Defects in infant vocalization have been proposed as an early sign of ASD that may exacerbate ASD development. However, the neural mechanisms associated with early communicative deficits in ASD are not known. Here, we expressed a constitutively active mutant of Rheb (RhebS16H), which is known to upregulate two ASD core pathways, mTOR complex 1 (mTORC1) and ERK1/2, in layer (L) 2/3 pyramidal neurons of the neocortex of mice of either sex. We found that cellular mosaic expression of RhebS16H in L2/3 pyramidal neurons altered the production of isolation calls from neonatal mice. This was accompanied by an expected misplacement of neurons and dendrite overgrowth, along with an unexpected increase in spine density and length, which was associated with increased excitatory synaptic activity. This contrasted with the known decrease in spine density in RhebS16H neurons of one-month-old mice. Reducing the levels of the actin crosslinking and adaptor protein filamin A (FLNA), known to be increased downstream of ERK1/2, attenuated dendrite overgrowth and fully restored spine properties, synaptic connectivity, and the production of pup isolation calls. These findings suggest that upper-layer cortical pyramidal neurons contribute to communicative deficits in a condition known to affect two core ASD pathways and that these mechanisms are regulated by FLNA.Significance Statement An infant's cry is a form of evolutionarily conserved social communication that is altered in ASD and has been proposed as an early sign of ASD. However, the neural substrate of early communicative deficits is not known. We show that cellular mosaic expression of a constitutively active Rheb, known to upregulate two core ASD pathways, selectively in upper-layer neocortical pyramidal neurons, alters the production of pup isolation calls and synaptic connectivity. These defects were prevented by reducing the expression of the adaptor protein FLNA. This underscores the importance of a specific neuronal substrate responsible for communicative deficits and its regulation by FLNA.

The mTOR pathway genes MTOR, Rheb, Depdc5, Pten, and Tsc1 have convergent and divergent impacts on cortical neuron development and function.

Brain somatic mutations in various components of the mTOR complex 1 (mTORC1) pathway have emerged as major causes of focal malformations of cortical development and intractable epilepsy. While these distinct gene mutations converge on excessive mTORC1 signaling and lead to common clinical manifestations, it remains unclear whether they cause similar cellular and synaptic disruptions underlying cortical network hyperexcitability. Here, we show that in utero activation of the mTORC1 activator genes, Rheb or MTOR, or biallelic inactivation of the mTORC1 repressor genes, Depdc5, Tsc1, or Pten in the mouse medial prefrontal cortex leads to shared alterations in pyramidal neuron morphology, positioning, and membrane excitability but different changes in excitatory synaptic transmission. Our findings suggest that, despite converging on mTORC1 signaling, mutations in different mTORC1 pathway genes differentially impact cortical excitatory synaptic activity, which may confer gene-specific mechanisms of hyperexcitability and responses to therapeutic intervention.

The mTOR pathway genes mTOR, Rheb, Depdc5, Pten, and Tsc1 have convergent and divergent impacts on cortical neuron development and function.

Brain somatic mutations in various components of the mTOR complex 1 (mTORC1) pathway have emerged as major causes of focal malformations of cortical development and intractable epilepsy. While these distinct gene mutations converge on excessive mTORC1 signaling and lead to common clinical manifestations, it remains unclear whether they cause similar cellular and synaptic disruptions underlying cortical network hyperexcitability. Here, we show that in utero activation of the mTORC1 activators, Rheb or mTOR, or biallelic inactivation of the mTORC1 repressors, Depdc5, Tsc1, or Pten in mouse medial prefrontal cortex leads to shared alterations in pyramidal neuron morphology, positioning, and membrane excitability but different changes in excitatory synaptic transmission. Our findings suggest that, despite converging on mTORC1 signaling, mutations in different mTORC1 pathway genes differentially impact cortical excitatory synaptic activity, which may confer gene-specific mechanisms of hyperexcitability and responses to therapeutic intervention.

Application of fast expectation-maximization microbial source tracking to discern fecal contamination in rivers exposed to low fecal inputs.

Community-based microbial source tracking (MST) can be used to determine fecal contamination from multiple sources in the aquatic environment. However, there is little scientific information on its application potential in water environmental management. Here, we compared SourceTracker and Fast Expectation-maximization Microbial Source Tracking (FEAST) performances on environmental water bodies exposed to low fecal pollution and evaluated treatment effects of fecal pollution in the watershed utilizing community-based MST. Our results showed that FEAST overall outperformed SourceTracker in sensitivity and stability, and was able to discern multi-source fecal contamination (mainly chicken feces) in ambient water bodies exposed to low fecal inputs. Consistent with our previous PCR/qPCR-based MST assays, FEAST analysis indicates that fecal pollution has been significantly mitigated through comprehensive environmental treatment by the local government. This study suggests that FEAST can be a powerful tool for accurately evaluating the contribution of multi-source fecal contamination in environmental water, facilitating environmental management.

Expression of 4E-BP1 in juvenile mice alleviates mTOR-induced neuronal dysfunction and epilepsy.

Hyperactivation of the mTOR pathway during foetal neurodevelopment alters neuron structure and function, leading to focal malformation of cortical development and intractable epilepsy. Recent evidence suggests a role for dysregulated cap-dependent translation downstream of mTOR signalling in the formation of focal malformation of cortical development and seizures. However, it is unknown whether modifying translation once the developmental pathologies are established can reverse neuronal abnormalities and seizures. Addressing these issues is crucial with regards to therapeutics because these neurodevelopmental disorders are predominantly diagnosed during childhood, when patients present with symptoms. Here, we report increased phosphorylation of the mTOR effector and translational repressor, 4E-BP1, in patient focal malformation of cortical development tissue and in a mouse model of focal malformation of cortical development. Using temporally regulated conditional gene expression systems, we found that expression of a constitutively active form of 4E-BP1 that resists phosphorylation by focal malformation of cortical development in juvenile mice reduced neuronal cytomegaly and corrected several neuronal electrophysiological alterations, including depolarized resting membrane potential, irregular firing pattern and aberrant expression of HCN4 ion channels. Further, 4E-BP1 expression in juvenile focal malformation of cortical development mice after epilepsy onset resulted in improved cortical spectral activity and decreased spontaneous seizure frequency in adults. Overall, our study uncovered a remarkable plasticity of the juvenile brain that facilitates novel therapeutic opportunities to treat focal malformation of cortical development-related epilepsy during childhood with potentially long-lasting effects in adults.

Muscle BDNF improves synaptic and contractile muscle strength in Kennedy's disease mice in a muscle-type specific manner.

KEY POINTS: Muscle-derived neurotrophic factors may offer therapeutic promise for treating neuromuscular diseases. We report that a muscle-derived neurotrophic factor, BDNF, rescues synaptic and muscle function in a muscle-type specific manner in mice modelling Kennedy's disease (KD). We also find that BDNF rescues select molecular mechanisms in slow and fast muscle that may underlie the improved cellular function. We also report for the first time that expression of BDNF, but not other members of the neurotrophin family, is perturbed in muscle from patients with KD. Given that muscle BDNF had divergent therapeutic effects that depended on muscle type, a combination of neurotrophic factors may optimally rescue neuromuscular function via effects on both pre- and postsynaptic function, in the face of disease. ABSTRACT: Deficits in muscle brain-derived neurotrophic factor (BDNF) correlate with neuromuscular deficits in mouse models of Kennedy's disease (KD), suggesting that restoring muscle BDNF might restore function. To test this possibility, transgenic mice expressing human BDNF in skeletal muscle were crossed with '97Q' KD mice. We found that muscle BDNF slowed disease, doubling the time between symptom onset and endstage. BDNF also improved expression of genes in muscle known to play key roles in neuromuscular function, including counteracting the expression of neonatal isoforms induced by disease. Intriguingly, BDNF's ameliorative effects differed between muscle types: synaptic strength was rescued only in slow-twitch muscle, while contractile strength was improved only in fast-twitch muscle. In sum, muscle BDNF slows disease progression, rescuing select cellular and molecular mechanisms that depend on fibre type. Muscle BDNF expression was also affected in KD patients, reinforcing its translational and therapeutic potential for treating this disorder.

Pre-clinical symptoms of SBMA may not be androgen-dependent: implications from two SBMA mouse models.

A distinguishing aspect of spinal and bulbar muscular atrophy (SBMA) is its androgen-dependence, possibly explaining why only males are clinically affected. This disease, which impairs neuromuscular function, is linked to a polyglutamine expansion mutation in the androgen receptor (AR). In mouse models of SBMA, motor dysfunction is associated with pronounced defects in neuromuscular transmission, including defects in evoked transmitter release (quantal content, QC) and fiber membrane excitability (based on the resting membrane potential, RMP). However, whether such defects are androgen-dependent is unknown. Thus, we recorded synaptic potentials intracellularly from adult muscle fibers of transgenic (Tg) AR97Q male mice castrated pre-symptomatically. Although castration largely protects both QC and the RMP of fibers, correlating with the protective effect of castration on motor function, significant deficits in QC and RMP remained. Surprisingly, comparable defects in QC and RMP were also observed in pre-symptomatic AR97Q males, indicating that such defects emerge early and are pre-clinical. Exposing asymptomatic Tg females to androgens also induces both motor dysfunction and comparable defects in QC and RMP. Notably, asymptomatic Tg females also showed significant deficits in QC and RMP, albeit less severe, supporting their pre-clinical nature, but also raising questions about the androgen-dependence of pre-clinical symptoms. In summary, current evidence indicates that disease progression depends on androgens, but early pathogenic events may be triggered by the mutant AR allele independent of androgens. Such early, androgen-independent disease mechanisms may also be relevant to females carrying the SBMA allele.

Defects in Neuromuscular Transmission May Underlie Motor Dysfunction in Spinal and Bulbar Muscular Atrophy.

UNLABELLED: Spinal and bulbar muscular atrophy (SBMA) in men is an androgen-dependent neuromuscular disease caused by expanded CAG repeats in the androgen receptor (AR). Whether muscle or motor neuron dysfunction or both underlies motor impairment in SBMA is unknown. Muscles of SBMA mice show significant contractile dysfunction, implicating them as a likely source of motor dysfunction, but whether disease also impairs neuromuscular transmission is an open question. Thus, we examined synaptic function in three well-studied SBMA mouse models-the AR97Q, knock-in (KI), and myogenic141 models-by recording in vitro miniature and evoked end-plate potentials (MEPPs and EPPs, respectively) intracellularly from adult muscle fibers. We found striking defects in neuromuscular transmission suggesting that toxic AR in SBMA impairs both presynaptic and postsynaptic mechanisms. Notably, SBMA causes neuromuscular synapses to become weak and muscles to become hyperexcitable in all three models. Presynaptic defects included deficits in quantal content, reduced size of the readily releasable pool, and impaired short-term facilitation. Postsynaptic defects included prolonged decay times for both MEPPs and EPPs, marked resistance to µ-conotoxin (a sodium channel blocker), and enhanced membrane excitability. Quantitative PCR revealed robust upregulation of mRNAs encoding neonatal isoforms of the AChR (γ-subunit) and the voltage-gated sodium channel (NaV1.5) in diseased adult muscles of all three models, consistent with the observed slowing of synaptic potentials and resistance to µ-conotoxin. These findings suggest that muscles of SBMA patients regress to an immature state that impairs neuromuscular function. SIGNIFICANCE STATEMENT: We have discovered that SBMA is accompanied by marked defects in neuromuscular synaptic transmission involving both presynaptic and postsynaptic mechanisms. For three different mouse models, we find that diseased synapses are weak, having reduced quantal content due to reductions in the size of the readily releasable pool and/or probability of release. Synaptic potentials in diseased adult fibers are slowed, explained by an aberrant upregulation of the neonatal isoform of the acetylcholine receptor. Diseased fibers also show marked resistance to µ-conotoxin, explained by an aberrant upregulation in the neonatal isoform of the sodium channel, and are hyperexcitable, reminiscent of myotonic dystrophy, showing anode-break action potentials. This work identifies several new molecular targets for recovering function in SBMA.

Contractile dysfunction in muscle may underlie androgen-dependent motor dysfunction in spinal bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is characterized by progressive muscle weakness linked to a polyglutamine expansion in the androgen receptor (AR). Current evidence indicates that mutant AR causes SBMA by acting in muscle to perturb its function. However, information about how muscle function is impaired is scant. One fundamental question is whether the intrinsic strength of muscles, an attribute of muscle independent of its mass, is affected. In the current study, we assess the contractile properties of hindlimb muscles in vitro from chronically diseased males of three different SBMA mouse models: a transgenic (Tg) model that broadly expresses a full-length human AR with 97 CAGs (97Q), a knock-in (KI) model that expresses a humanized AR containing a CAG expansion in the first exon, and a Tg myogenic model that overexpresses wild-type AR only in skeletal muscle fibers. We found that hindlimb muscles in the two Tg models (97Q and myogenic) showed marked losses in their intrinsic strength and resistance to fatigue, but were minimally affected in KI males. However, diseased muscles of all three models showed symptoms consistent with myotonic dystrophy type 1, namely, reduced resting membrane potential and deficits in chloride channel mRNA. These data indicate that muscle dysfunction is a core feature of SBMA caused by at least some of the same pathogenic mechanisms as myotonic dystrophy. Thus mechanisms controlling muscle function per se independent of mass are prime targets for SBMA therapeutics.

Age-dependent contribution of P/Q- and R-type Ca2+ channels to neuromuscular transmission in lethargic mice.

ß-Subunits of voltage-gated calcium channels (VGCCs) regulate assembly and membrane localization of the pore-forming α1-subunit and strongly influence channel function. ß4-Subunits normally coassociate with α1A-subunits which comprise P/Q-type (Cav2.1) VGCCs. These control acetylcholine (ACh) release at adult mammalian neuromuscular junctions (NMJs). The naturally occurring lethargic (lh) mutation of the ß4-subunit in mice causes loss of the α1-binding site, possibly affecting P/Q-type channel expression or function, and thereby ACh release. End-plate potentials and miniature end-plate potentials were recorded at hemidiaphragm NMJs of 5-7-week and 3-5-month-old lh and wild-type (wt) mice. Sensitivity to antagonists of P/Q- [ω-agatoxin IVA (ω-Aga-IVA)], L- (nimodipine), N- (ω-conotoxin GVIA), and R-type [C192H274N52O60S7 (SNX-482)] VGCCs was compared in juvenile and adult lh and wt mice. Quantal content (m) of adult, but not juvenile, lh mice was reduced compared to wt. ω-Aga-IVA (~60%) and SNX-482 (~ 45%) significantly reduced m in adult lh mice. Only Aga-IVA affected wt adults. In juvenile lh mice, ω-Aga-IVA and SNX-482 decreased m by >75% and ~20%, respectively. Neither ω-conotoxin GVIA nor nimodipine affected ACh release in any group. Immunolabeling revealed α1E and α1A, ß1, and ß3 staining at adult lh, but not wt NMJs. Therefore, in lh mice, when the ß-subunit that normally coassociates with α1A to form P/Q channels is missing, P/Q-type channels partner with other ß-subunits. However, overall participation of P/Q-type channels is reduced and compensated for by R-type channels. R-type VGCC participation is age-dependent, but is less effective than P/Q-type at sustaining NMJ function.

Reversed synaptic effects of hypocretin and NPY mediated by excitatory GABA-dependent synaptic activity in developing MCH neurons.

In mature neurons, GABA is the primary inhibitory neurotransmitter. In contrast, in developing neurons, GABA exerts excitatory actions, and in some neurons GABA-mediated excitatory synaptic activity is more prevalent than glutamate-mediated excitation. Hypothalamic neuropeptides that modulate cognitive arousal and energy homeostasis, hypocretin/orexin and neuropeptide Y (NPY), evoked reversed effects on synaptic actions that were dependent on presynaptic GABA release onto melanin-concentrating hormone (MCH) neurons. MCH neurons were identified by selective green fluorescent protein (GFP) expression in transgenic mice. In adults, hypocretin increased GABA release leading to reduced excitation. In contrast, in the developing brain as studied here with analysis of miniature excitatory postsynaptic currents, paired-pulse ratios, and evoked potentials, hypocretin acted presynaptically to enhance the excitatory actions of GABA. The ability of hypocretin to enhance GABA release increases inhibition in adult neurons but paradoxically enhances excitation in developing MCH neurons. In contrast, NPY attenuation of GABA release reduced inhibition in mature neurons but enhanced inhibition during development by attenuating GABA excitation. Both hypocretin and NPY also evoked direct actions on developing MCH neurons. Hypocretin excited MCH cells by activating a sodium-calcium exchanger and by reducing potassium currents; NPY reduced activity by increasing an inwardly rectifying potassium current. These data for the first time show that both hypocretin and NPY receptors are functional presynaptically during early postnatal hypothalamic development and that both neuropeptides modulate GABA actions during development with a valence of enhanced excitation or inhibition opposite to that of the adult state, potentially allowing neuropeptide modulation of use-dependent synapse stabilization.

Modulation of NMDA receptor function by inhibition of D-amino acid oxidase in rodent brain.

Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are similar to those seen in schizophrenia have led to the current hypothesis that schizophrenia might result from NMDA receptor hypofunction. Inhibition of D-amino acid oxidase (DAAO), the enzyme responsible for degradation of D-serine, should lead to increased levels of this co-agonist at the NMDA receptor, and thereby provide a therapeutic approach to schizophrenia. We have profiled some of the preclinical biochemical, electrophysiological, and behavioral consequences of administering potent and selective inhibitors of DAAO to rodents to begin to test this hypothesis. Inhibition of DAAO activity resulted in a significant dose and time dependent increase in D-serine only in the cerebellum, although a time delay was observed between peak plasma or brain drug concentration and cerebellum D-serine response. Pharmacokinetic/pharmacodynamic (PK/PD) modeling employing a mechanism-based indirect response model was used to characterize the correlation between free brain drug concentration and D-serine accumulation. DAAO inhibitors had little or no activity in rodent models considered predictive for antipsychotic activity. The inhibitors did, however, affect cortical activity in the Mescaline-Induced Scratching model, produced a modest but significant increase in NMDA receptor-mediated synaptic currents in primary neuronal cultures from rat hippocampus, and resulted in a significant increase in evoked hippocampal theta rhythm, an in vivo electrophysiological model of hippocampal activity. These findings demonstrate that although DAAO inhibition did not cause a measurable increase in D-serine in forebrain, it did affect hippocampal and cortical activity, possibly through augmentation of NMDA receptor-mediated currents.

Nicotine excites hypothalamic arcuate anorexigenic proopiomelanocortin neurons and orexigenic neuropeptide Y neurons: similarities and differences.

Two of the biggest health problems facing us today are addiction to nicotine and the increased prevalence of obesity. Interestingly, nicotine attenuates obesity, but the underlying mechanism is not clear. Here we address the hypothesis that if weight-reducing actions of nicotine are mediated by anorexigenic proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus, nicotine should excite these cells. Nicotine at concentrations similar to those found in smokers, 100-1,000 nM, excited POMC cells by mechanisms based on increased spike frequency, depolarization of membrane potential, and opening of ion channels. This was mediated by activation of both α7 and α4ß2 nicotinic receptors; by itself, this nicotine-mediated excitation could explain weight loss caused by nicotine. However, in control experiments nicotine also excited the orexigenic arcuate nucleus neuropeptide Y (NPY) cells. Nicotine exerted similar actions on POMC and NPY cells, with a slightly greater depolarizing action on POMC cells. Immunocytochemistry revealed cholinergic axons terminating on both cell types. Nicotine actions were direct in both cell types, with nicotine depolarizing the membrane potentials and reducing input resistance. We found no differences in the relative desensitization to nicotine between POMC and NPY neurons. Nicotine inhibited excitatory synaptic activity recorded in NPY, but not POMC, cells. Nicotine also excited hypocretin/orexin neurons that enhance cognitive arousal, but the responses were smaller than in NPY or POMC cells. Together, these results indicate that nicotine has a number of similar actions, but also a few different actions, on POMC and NPY neurons that could contribute to the weight loss associated with smoking.

Morphometric characterization of the neuromuscular junction of rodents intoxicated with 2,4-dithiobiuret: evidence that nerve terminal recycling processes contribute to muscle weakness.

2,4-Dithiobiuret (DTB) causes ascending motor weakness when given chronically to rodents. In muscles of animals with DTB-induced weakness, quantal release of acetylcholine (ACh) is impaired. We examined in detail the structural changes that occurred at neuromuscular junctions and their associated Schwann cells of extensor digitorum longus (EDL) muscles of male rats treated with DTB to the onset of muscle weakness, 5-8 days. Our objective was to assess the involvement of the Schwann cells and to determine the most likely primary targets of DTB. At the onset of muscle weakness, nerve terminals exhibited some enlarged regions, but did not sprout. Terminal Schwann cells became flatter and expanded to cover most of the endplate. The extent of invasion of the synaptic cleft by Schwann cell processes was not significantly different from controls; extension of Schwann cell sprouts away from the junction was not seen. Thus, the morphology of the Schwann cells, although clearly affected by DTB, does not suggest that they contribute directly to the physiological defects of DTB-treated terminals. Abnormal tubulovesicular structures or tangles of neurofilaments were clustered in the centers of about 25% of treated terminals. Fewer synaptic vesicles occupied the region opposite the postsynaptic folds. Vesicle volumes were variable and included some very large vesicles, corresponding with the variable MEPP amplitudes reported previously for terminals of DTB-treated rodents. The postsynaptic area stained by rhodamine-labeled alpha-bungarotoxin expanded with terminal swelling, apparently by unpleating of the postsynaptic folds. No loss of ACh receptors or spread of ACh receptors beyond terminal boundaries was detected. Morphometric data are consistent with the conclusion that DTB affects, either directly or indirectly, vesicular release of ACh and the subsequent vesicular recycling process.

Impairment of synaptic vesicle exocytosis and recycling during neuromuscular weakness produced in mice by 2,4-dithiobiuret.

Chronic treatment of rodents with 2,4-dithiobiuret (DTB) induces a neuromuscular syndrome of flaccid muscle weakness that mimics signs seen in several human neuromuscular disorders such as congenital myasthenic syndromes, botulism, and neuroaxonal dystrophy. DTB-induced muscle weakness results from a reduction of acetylcholine (ACh) release by mechanisms that are not yet clear. The objective of this study was to determine if altered release of ACh during DTB-induced muscle weakness was due to impairments of synaptic vesicle exocytosis, endocytosis, or internal vesicular processing. We examined motor nerve terminals in the triangularis sterni muscles of DTB-treated mice at the onset of muscle weakness. Uptake of FM1-43, a fluorescent marker for endocytosis, was reduced to approximately 60% of normal after either high-frequency nerve stimulation or K(+) depolarization. Terminals ranged from those with nearly normal fluorescence ("bright terminals") to terminals that were poorly labeled ("dim terminals"). Ultrastructurally, the number of synaptic vesicles that were labeled with horseradish peroxidase (HRP) was also reduced by DTB to approximately 60%; labeling among terminals was similarly variable. A subset of DTB-treated terminals having abnormal tubulovesicular profiles in their centers did not respond to stimulation with increased uptake of HRP and may correspond to dim terminals. Two findings suggest that posttetanic "slow endocytosis" remained qualitatively normal: the rate of this type of endocytosis as measured with FM1-43 did not differ from normal, and HRP was observed in organelles associated with this pathway- coated vesicles, cisternae, as well as synaptic vesicles but not in the tubulovesicular profiles. In DTB-treated bright terminals, end-plate potential (EPP) amplitudes were decreased, and synaptic depression in response to 15-Hz stimulation was increased compared with those of untreated mice; in dim terminals, EPPs were not observed during block with D-tubocurarine. Nerve-stimulation-induced unloading of FM1-43 was slower and less complete than normal in bright terminals, did not occur in dim terminals, and was not enhanced by alpha-latrotoxin. Collectively, these results indicate that the size of the recycling vesicle pool is reduced in nerve terminals during DTB-induced muscle weakness. The mechanisms by which this reduction occurs are not certain, but accumulated evidence suggests that they may include defects in either or both exocytosis and internal vesicular processing.