Identification of a rare MET variant in a familial case of extramammary Paget's disease.

Extramammary Paget's disease (EMPD) is an intraepithelial adenocarcinoma that primarily affects the genital and axillary areas in elderly individuals. A limited number of paired familial EMPD cases (i.e., parent-offspring, siblings) have been reported, whereas the genetics of these cases have not yet been adequately studied. We report the first familial case of EMPD involving three affected siblings. The tumour-only multi-gene panel testing using surgical specimens revealed a heterozygous c.2997A>C (p.Glu999Asp) nonsynonymous variant in the proto-oncogene MET (NM_000245.4) in the three affected siblings. The germline multi-gene panel testing using peripheral blood lymphocytes revealed the same missense MET variant in all five family members, including the two asymptomatic offspring (51 and 37 years of age). The MET variant we identified could be involved in EMPD carcinogenesis. Further genomic analyses of familial cases of EMPD are warranted to validate the pathogenic relevance of MET variants in EMPD development.

Anterior cingulate cross-hemispheric inhibition via the claustrum resolves painful sensory conflict.

The anterior cingulate cortex (ACC) responds to noxious and innocuous sensory inputs, and integrates them to coordinate appropriate behavioral reactions. However, the role of the projections of ACC neurons to subcortical areas and their influence on sensory processing are not fully investigated. Here, we identified that ACC neurons projecting to the contralateral claustrum (ACC→contraCLA) preferentially respond to contralateral mechanical sensory stimulation. These sensory responses were enhanced during attending behavior. Optogenetic activation of ACC→contraCLA neurons silenced pyramidal neurons in the contralateral ACC by recruiting local circuit fast-spiking interneuron activation via an excitatory relay in the CLA. This circuit activation suppressed withdrawal behavior to mechanical stimuli ipsilateral to the ACC→contraCLA neurons. Chemogenetic silencing showed that the cross-hemispheric circuit has an important role in the suppression of contralateral nociceptive behavior during sensory-driven attending behavior. Our findings identify a cross-hemispheric cortical-subcortical-cortical arc allowing the brain to give attentional priority to competing innocuous and noxious inputs.

Anterograde trans-neuronal labeling of striatal interneurons in relation to dopamine neurons in the substantia nigra pars compacta.

Recent advances in neural tracing have unveiled numerous neural circuits characterized by brain region and cell type specificity, illuminating the underpinnings of specific functions and behaviors. Dopaminergic (DA) neurons in the midbrain are highly heterogeneous in terms of gene and protein expression and axonal projections. Different cell types within the substantia nigra pars compacta (SNc) tend to project to the striatum in a cell-type-dependent manner characterized by specific topography. Given the wide and dense distribution of DA axons, coupled with a combination of synaptic and volume transmission, it remains unclear how DA release is spatially and temporally regulated, to appropriately achieve specific behaviors and functions. Our hypothesis posits that hidden rules governing synapse formation between pre-synaptic DA neuron types and striatal neuron types may modulate the effect of DA at a single-cell level. To address this conjecture, we employed adeno-associated virus serotype 1 (AAV1) to visualize the neural circuitry of DA neurons. AAV1 has emerged as a potent anatomical instrument capable of labeling and visualizing pre- and post-synaptic neurons simultaneously through anterograde trans-synaptic labeling. First, AAV1-Cre was injected into the SNc, resulting in Cre expression in both medium spiny neurons and interneurons in the striatum. Due to the potential occurrence of the retrograde transfer of AAV1, only striatal interneurons were considered for trans-synaptic or trans-neuronal labeling. Interneuron types expressing parvalbumin, choline acetyltransferase, somatostatin, or nitrogen oxide synthase exhibited Cre expression. Using a combination of AAV1-Cre and Cre-driven fluorophore expressing AAVs, striatal interneurons and the axons originating from the SNc were visualized in distinct colors. Using immunofluorescence against neurotransmitter transporters, almost all axons in the striatum visualized using this approach were confirmed to be dopaminergic. Moreover, individual DA axons established multiple appositions on the somata and proximal dendrites of interneurons. This finding suggests that irrespective of the extensive and widespread axonal arborization of DA neurons, a particular DA neuron may exert a significant influence on specific interneurons. Thus, AAV1-based labeling of the DA system can be a valuable tool to uncover the concealed rules governing these intricate relationships.

IκB kinase phosphorylates cytoplasmic TDP-43 and promotes its proteasome degradation.

Cytoplasmic aggregation of TDP-43 in neurons is a pathological feature common to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We demonstrate that the IκB kinase (IKK) complex promotes the degradation of cytoplasmic TDP-43 through proteasomes. While IKKß is a major factor in TDP-43 degradation, IKKα acts as a cofactor, and NEMO functions as a scaffold for the recruitment of TDP-43 to the IKK complex. Furthermore, we identified IKKß-induced phosphorylation sites of TDP-43 and found that phosphorylation at Thr8 and Ser92 is important for the reduction of TDP-43 by IKK. TDP-43 phosphorylation at Ser92 was detected in a pattern different from that of C-terminal phosphorylation in the pathological inclusion of ALS. IKKß was also found to significantly reduce the expression level and toxicity of the disease-causing TDP-43 mutation. Finally, the favorable effect of IKKß on TDP-43 aggregation was confirmed in the hippocampus of mice. IKK and the N-terminal phosphorylation of TDP-43 are potential therapeutic targets for ALS and FTLD.

Enhancement of Haloperidol-Induced Catalepsy by GPR143, an L-Dopa Receptor, in Striatal Cholinergic Interneurons.

Dopamine neurons play crucial roles in pleasure, reward, memory, learning, and fine motor skills and their dysfunction is associated with various neuropsychiatric diseases. Dopamine receptors are the main target of treatment for neurologic and psychiatric disorders. Antipsychotics that antagonize the dopamine D2 receptor (DRD2) are used to alleviate the symptoms of these disorders but may also sometimes cause disabling side effects such as parkinsonism (catalepsy in rodents). Here we show that GPR143, a G-protein-coupled receptor for L-3,4-dihydroxyphenylalanine (L-DOPA), expressed in striatal cholinergic interneurons enhances the DRD2-mediated side effects of haloperidol, an antipsychotic agent. Haloperidol-induced catalepsy was attenuated in male Gpr143 gene-deficient (Gpr143-/y ) mice compared with wild-type (Wt) mice. Reducing the endogenous release of L-DOPA and preventing interactions between GPR143 and DRD2 suppressed the haloperidol-induced catalepsy in Wt mice but not Gpr143-/y mice. The phenotypic defect in Gpr143-/y mice was mimicked in cholinergic interneuron-specific Gpr143-/y (Chat-cre;Gpr143flox/y ) mice. Administration of haloperidol increased the phosphorylation of ribosomal protein S6 at Ser240/244 in the dorsolateral striatum of Wt mice but not Chat-cre;Gpr143flox/y mice. In Chinese hamster ovary cells stably expressing DRD2, co-expression of GPR143 increased cell surface expression level of DRD2, and L-DOPA application further enhanced the DRD2 surface expression. Shorter pauses in cholinergic interneuron firing activity were observed after intrastriatal stimulation in striatal slice preparations from Chat-cre;Gpr143flox/y mice compared with those from Wt mice. Together, these findings provide evidence that GPR143 regulates DRD2 function in cholinergic interneurons and may be involved in parkinsonism induced by antipsychotic drugs.

Balancing risk-return decisions by manipulating the mesofrontal circuits in primates.

Decision-making is always coupled with some level of risk, with more pathological forms of risk-taking decisions manifesting as gambling disorders. In macaque monkeys trained in a high risk-high return (HH) versus low risk-low return (LL) choice task, we found that the reversible pharmacological inactivation of ventral Brodmann area 6 (area 6V) impaired the risk dependency of decision-making. Selective optogenetic activation of the mesofrontal pathway from the ventral tegmental area (VTA) to the ventral aspect of 6V resulted in stronger preference for HH, whereas activation of the pathway from the VTA to the dorsal aspect of 6V led to LL preference. Finally, computational decoding captured the modulations of behavioral preference. Our results suggest that VTA inputs to area 6V determine the decision balance between HH and LL.

Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity.

Neurodevelopmental disorders, such as intellectual disability (ID), epilepsy, and autism, involve altered synaptic transmission and plasticity. Functional characterization of their associated genes is vital for understanding physio-pathological brain functions. LGI3 is a recently recognized ID-associated gene encoding a secretory protein related to an epilepsy-gene product, LGI1. Here, we find that LGI3 is uniquely secreted from oligodendrocytes in the brain and enriched at juxtaparanodes of myelinated axons, forming nanoscale subclusters. Proteomic analysis using epitope-tagged Lgi3 knockin mice shows that LGI3 uses ADAM23 as a receptor and selectively co-assembles with Kv1 channels. A lack of Lgi3 in mice disrupts juxtaparanodal clustering of ADAM23 and Kv1 channels and suppresses Kv1-channel-mediated short-term synaptic plasticity. Collectively, this study identifies an extracellular organizer of juxtaparanodal Kv1 channel clustering for finely tuned synaptic transmission. Given the defective secretion of the LGI3 missense variant, we propose a molecular pathway, the juxtaparanodal LGI3-ADAM23-Kv1 channel, for understanding neurodevelopmental disorders.

Two parabrachial Cck neurons involved in the feedback control of thirst or salt appetite.

Thirst and salt appetite are temporarily suppressed after water and salt ingestion, respectively, before absorption; however, the underlying neural mechanisms remain unclear. The parabrachial nucleus (PBN) is the relay center of ingestion signals from the digestive organs. We herein identify two distinct neuronal populations expressing cholecystokinin (Cck) mRNA in the lateral PBN that are activated in response to water and salt intake, respectively. The two Cck neurons in the dorsal-lateral compartment of the PBN project to the median preoptic nucleus and ventral part of the bed nucleus of the stria terminalis, respectively. The optogenetic stimulation of respective Cck neurons suppresses thirst or salt appetite under water- or salt-depleted conditions. The combination of optogenetics and in vivo Ca2+ imaging during ingestion reveals that both Cck neurons control GABAergic neurons in their target nuclei. These findings provide the feedback mechanisms for the suppression of thirst and salt appetite after ingestion.

Cold-induced suspension and resetting of Ca2+ and transcriptional rhythms in the suprachiasmatic nucleus neurons.

Does the circadian clock keep running under such hypothermic states as daily torpor and hibernation? This fundamental question has been a research subject for decades but has remained unsettled. We addressed this subject by monitoring the circadian rhythm of clock gene transcription and intracellular Ca2+ in the neurons of the suprachiasmatic nucleus (SCN), master circadian clock, in vitro under a cold environment. We discovered that the transcriptional and Ca2+ rhythms are maintained at 22°C-28°C, but suspended at 15°C, accompanied by a large Ca2+ increase. Rewarming instantly resets the Ca2+ rhythms, while transcriptional rhythms reach a stable phase after the transient state and recover their phase relationship with the Ca2+ rhythm. We conclude that SCN neurons remain functional under moderate hypothermia but stop ticking in deep hypothermia and that the rhythms reset after rewarming. These data also indicate that stable Ca2+ oscillation precedes clock gene transcriptional rhythms in SCN neurons.

Change detection in the primate auditory cortex through feedback of prediction error signals.

Although cortical feedback signals are essential for modulating feedforward processing, no feedback error signal across hierarchical cortical areas has been reported. Here, we observed such a signal in the auditory cortex of awake common marmoset during an oddball paradigm to induce auditory duration mismatch negativity. Prediction errors to a deviant tone presentation were generated as offset calcium responses of layer 2/3 neurons in the rostral parabelt (RPB) of higher-order auditory cortex, while responses to non-deviant tones were strongly suppressed. Within several hundred milliseconds, the error signals propagated broadly into layer 1 of the primary auditory cortex (A1) and accumulated locally on top of incoming auditory signals. Blockade of RPB activity prevented deviance detection in A1. Optogenetic activation of RPB following tone presentation nonlinearly enhanced A1 tone response. Thus, the feedback error signal is critical for automatic detection of unpredicted stimuli in physiological auditory processing and may serve as backpropagation-like learning.

Fibroblast growth factor 18 stimulates the proliferation of hepatic stellate cells, thereby inducing liver fibrosis.

Liver fibrosis results from chronic liver injury triggered by factors such as viral infection, excess alcohol intake, and lipid accumulation. However, the mechanisms underlying liver fibrosis are not fully understood. Here, we demonstrate that the expression of fibroblast growth factor 18 (Fgf18) is elevated in mouse livers following the induction of chronic liver fibrosis models. Deletion of Fgf18 in hepatocytes attenuates liver fibrosis; conversely, overexpression of Fgf18 promotes liver fibrosis. Single-cell RNA sequencing reveals that overexpression of Fgf18 in hepatocytes results in an increase in the number of Lrat+ hepatic stellate cells (HSCs), thereby inducing fibrosis. Mechanistically, FGF18 stimulates the proliferation of HSCs by inducing the expression of Ccnd1. Moreover, the expression of FGF18 is correlated with the expression of profibrotic genes, such as COL1A1 and ACTA2, in human liver biopsy samples. Thus, FGF18 promotes liver fibrosis and could serve as a therapeutic target to treat liver fibrosis.

Anatomical identification of a corticocortical top-down recipient inhibitory circuitry by enhancer-restricted transsynaptic tracing.

Despite the importance of postsynaptic inhibitory circuitry targeted by mid/long-range projections (e.g., top-down projections) in cognitive functions, its anatomical properties, such as laminar profile and neuron type, are poorly understood owing to the lack of efficient tracing methods. To this end, we developed a method that combines conventional adeno-associated virus (AAV)-mediated transsynaptic tracing with a distal-less homeobox (Dlx) enhancer-restricted expression system to label postsynaptic inhibitory neurons. We called this method "Dlx enhancer-restricted Interneuron-SpECific transsynaptic Tracing" (DISECT). We applied DISECT to a top-down corticocortical circuit from the secondary motor cortex (M2) to the primary somatosensory cortex (S1) in wild-type mice. First, we injected AAV1-Cre into the M2, which enabled Cre recombinase expression in M2-input recipient S1 neurons. Second, we injected AAV1-hDlx-flex-green fluorescent protein (GFP) into the S1 to transduce GFP into the postsynaptic inhibitory neurons in a Cre-dependent manner. We succeeded in exclusively labeling the recipient inhibitory neurons in the S1. Laminar profile analysis of the neurons labeled via DISECT indicated that the M2-input recipient inhibitory neurons were distributed in the superficial and deep layers of the S1. This laminar distribution was aligned with the laminar density of axons projecting from the M2. We further classified the labeled neuron types using immunohistochemistry and in situ hybridization. This post hoc classification revealed that the dominant top-down M2-input recipient neuron types were somatostatin-expressing neurons in the superficial layers and parvalbumin-expressing neurons in the deep layers. These results demonstrate that DISECT enables the investigation of multiple anatomical properties of the postsynaptic inhibitory circuitry.

Highly sensitive visual restoration and protection via ectopic expression of chimeric rhodopsin in mice.

Photoreception requires amplification by mammalian rhodopsin through G protein activation, which requires a visual cycle. To achieve this in retinal gene therapy, we incorporated human rhodopsin cytoplasmic loops into Gloeobacter rhodopsin, thereby generating Gloeobacter and human chimeric rhodopsin (GHCR). In a murine model of inherited retinal degeneration, we induced retinal GHCR expression by intravitreal injection of a recombinant adeno-associated virus vector. Retinal explant and visual thalamus electrophysiological recordings, behavioral tests, and histological analysis showed that GHCR restored dim-environment vision and prevented the progression of retinal degeneration. Thus, GHCR may be a potent clinical tool for the treatment of retinal disorders.

Clinicopathological significance of androgen receptor expression in extramammary Paget disease: An analysis of 92 patients.

Extramammary Paget disease (EMPD) is a rare cutaneous malignant neoplasm arising in apocrine gland-rich areas. Although - like normal apocrine glands - EMPD frequently expresses androgen receptor (AR), the clinical significance of AR expression remains unclear. The present study investigated the clinicopathological impact of AR expression in EMPD. Immunohistochemistry for AR was performed in a retrospective cohort of 92 EMPD patients with 108 EMPD lesions, including 102 primary lesions, five lymph node [LN] metastases and one local recurrence. The total AR staining score was calculated as staining intensity score (IS 0-3) × positive-cell percentage score (PS 1-4). Expression levels were graded as Grade 1 (scores 0 and 1), Grade 2 (scores 2-4), and Grade 3 (scores 6-12). Higher expression grade was correlated with tumor thickness (P = 0.011), LN metastasis (P = 0.008), and higher EMPD stage (P = 0.023). Grade 1 EMPDs did not invade into the dermis and did not generate metastatic and/or recurrent lesions, whereas only Grade 2 or 3 EMPDs did so. AR expression in invasive components was significantly higher (P = 0.023) than in non-invasive components remaining within the epidermis. AR expression was further elevated in metastatic and/or recurrent lesions relative to locally invasive lesions (P = 0.014). These results clearly indicate that increased AR expression is associated with malignant progression of EMPD and that androgen blockade might be an effective therapy. Furthermore, AR expression assessed by immunohistochemistry may have potential for prediction of LN metastasis and local recurrence in EMPD.

Hippocampal ensemble dynamics and memory performance are modulated by respiration during encoding.

During offline brain states, such as sleep and memory consolidation, respiration coordinates hippocampal activity. However, the role of breathing during online memory traces remains unclear. Here, we show that respiration can be recruited during online memory encoding. Optogenetic manipulation was used to control activation of the primary inspiratory rhythm generator PreBötzinger complex (PreBötC) in transgenic mice. When intermittent PreBötC-induced apnea covered the object exploration time during encoding, novel object detection was impaired. Moreover, the mice did not exhibit freezing behavior during presentation of fear-conditioned stimuli (CS+) when PreBötC-induced apnea occurred at the exact time of encoding. This apnea did not evoke changes in CA3 cell ensembles between presentations of CS+ and conditioned inhibition (CS-), whereas in normal breathing, CS+ presentations produced dynamic changes. Our findings demonstrate that components of central respiratory activity (e.g., frequency) during online encoding strongly contribute to shaping hippocampal ensemble dynamics and memory performance.

Chemogenetic dissection of a prefrontal-hypothalamic circuit for socially subjective reward valuation in macaques.

The value of one's own reward is affected by the reward of others, serving as a source for envy. However, it is not known which neural circuits mediate such socially subjective value modulation. Here, we chemogenetically dissected the circuit from the medial prefrontal cortex (MPFC) to the lateral hypothalamus (LH) while male macaques were presented with visual stimuli that concurrently signaled the prospects of one's own and others' rewards. We found that functional disconnection between the MPFC and LH rendered animals significantly less susceptible to others' but not one's own reward prospects. In parallel with this behavioral change, inter-areal coordination, as indexed by coherence and Granger causality, decreased primarily in the delta and theta bands. These findings demonstrate that the MPFC-to-LH circuit plays a crucial role in carrying information about upcoming other-rewards for subjective reward valuation in social contexts.

Starburst amacrine cells amplify optogenetic visual restoration through gap junctions.

Ectopic induction of optogenetic actuators, such as channelrhodopsin, is a promising approach to restoring vision in the degenerating retina. However, the cell type-specific response of ectopic photoreception has not been well understood. There are limits to obtaining efficient gene expression in a specifically targeted cell population by a transgenic approach. In the present study, we established a murine model with high efficiency of gene induction to retinal ganglion cells (RGCs) and amacrine cells using an improved tetracycline transactivator-operator bipartite system (KENGE-tet system). To investigate the cell type-specific visual restorative effect, we expressed the channelrhodopsin gene into RGCs and amacrine cells using the KENGE-tet system. As a result, enhancement in the visual restorative effect was observed to RGCs and starburst amacrine cells. In conclusion, a photoresponse from amacrine cells may enhance the maintained response of RGCs and further increase or improve the visual restorative effect.

Voltage-gated calcium channel subunit α2δ-1 in spinal dorsal horn neurons contributes to aberrant excitatory synaptic transmission and mechanical hypersensitivity after peripheral nerve injury.

Neuropathic pain, an intractable pain symptom that occurs after nerve damage, is caused by the aberrant excitability of spinal dorsal horn (SDH) neurons. Gabapentinoids, the most commonly used drugs for neuropathic pain, inhibit spinal calcium-mediated neurotransmitter release by binding to α2δ-1, a subunit of voltage-gated calcium channels, and alleviate neuropathic pain. However, the exact contribution of α2δ-1 expressed in SDH neurons to the altered synaptic transmission and mechanical hypersensitivity following nerve injury is not fully understood. In this study, we investigated which types of SDH neurons express α2δ-1 and how α2δ-1 in SDH neurons contributes to the mechanical hypersensitivity and altered spinal synaptic transmission after nerve injury. Using in situ hybridization technique, we found that Cacna2d1, mRNA coding α2δ-1, was mainly colocalized with Slc17a6, an excitatory neuronal marker, but not with Slc32a1, an inhibitory neuronal marker in the SDH. To investigate the role of α2δ-1 in SDH neurons, we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system and showed that SDH neuron-specific ablation of Cacna2d1 alleviated mechanical hypersensitivity following nerve injury. We further found that excitatory post-synaptic responses evoked by electrical stimulation applied to the SDH were significantly enhanced after nerve injury, and that these enhanced responses were significantly decreased by application of mirogabalin, a potent α2δ-1 inhibitor, and by SDH neuron-specific ablation of Cacna2d1. These results suggest that α2δ-1 expressed in SDH excitatory neurons facilitates spinal nociceptive synaptic transmission and contributes to the development of mechanical hypersensitivity after nerve injury.