Expression, mutation, and methylation of cereblon-pathway genes at pre- and post-lenalidomide treatment in multiple myeloma.

Cereblon (CRBN) is a target for immunomodulatory drugs. This study investigated the prognostic value of the expression of CRBN-pathway genes on the clinical relevance of lenalidomide (Len) treatment and evaluated the levels of CRBN-binding proteins and mutations in these genes after Len treatment. Forty-eight primary multiple myeloma cells were collected prior to treatment with Len and dexamethasone (Ld) and 25 paired samples were obtained post-Ld therapy. These tumor cells were used to determine the expression and mutated forms of the CRBN-pathway genes. Following normalization with CRBN levels, there was a significantly reduced IKZF1/CRBN ratio in samples that responded poorly to Ld therapy. Moreover, patients with low ratios of IKZF1/CRBN showed a significantly shorter progression-free survival (PFS) and overall survival (OS) than those with higher ratios. However, patients with high ratios of KPNA2/CRBN showed a significantly shorter PFS and OS than patients with lower ratios. Of the 25 paired samples analyzed, most samples showed a reduction in the expression of CRBN and an increase in IKZF1 gene expression. No mutations were observed in CRBN, IKZF1, or CUL4A genes in the post-Ld samples. In conclusion, a decreased expression of IKZF1 and increased expression of KPNA2 compared to that of CRBN mRNA predicts poor outcomes of Ld therapy.

Multizonal Cerebellar Influence Over Sensorimotor Areas of the Rat Cerebral Cortex.

The cerebral cortex requires cerebellar input for optimizing sensorimotor processing. However, how the sensorimotor cortex uses cerebellar information is far from understood. One critical and unanswered question is how cerebellar functional entities (zones or modules) are connected to distinct parts of the sensorimotor cortices. Here, we utilized retrograde transneuronal infection of rabies virus (RABV) to study the organization of connections from the cerebellar cortex to M1, M2, and S1 of the rat cerebral cortex. RABV was co-injected with cholera toxin ß-subunit (CTb) into each of these cortical regions and a survival time of 66-70 h allowed for third-order retrograde RABV infection of Purkinje cells. CTb served to identify the injection site. RABV+ Purkinje cells throughout cerebellar zones were identified by reference to the cerebellar zebrin pattern. All injections, including those into S1, resulted in multiple, zonally arranged, strips of RABV+ Purkinje cells. M1 injections were characterized by input from Purkinje cells in the vermal X-zone, medial paravermis (C1- and Cx-zones), and lateral hemisphere (D2-zone); M2 receives input from D2- and C3-zones; connections to S1 originate from X-, Cx-, C3-, and D2-zones. We hypothesize that individual domains of the sensorimotor cortex require information from a specific combination of cerebellar modules.

Cholinergic interneurons in the rat striatum modulate substitution of habits.

Behavioural flexibility is crucial for adaptive behaviour, and recent evidence suggests that cholinergic interneurons of the striatum play a distinct role. Previous studies of cholinergic function have focused on strategy switching by the dorsomedial or ventral striatum. We here investigated whether cholinergic interneurons in the dorsolateral striatum play a similar role at the level of switching of habitual responses. Because the dorsolateral striatum is particularly involved in habitual responding, we developed a habit substitution task that involved switching habitual lever-press responses to one side to another. We first measured the effect of cholinergic activation in the dorsolateral striatum on this task. Chemogenetic activation of cholinergic interneurons caused an increase in the response rate for the substituted response that was significantly greater than the increase normally seen in control animals. The increase was due to burst-like responses with shorter inter-press intervals. However, there was no effect on inhibiting the old habit, or on habitual responding that did not require a switch. There was also no effect on lever-press performance and its reversal before lever-press responses became habitual. Conversely, neurochemically specific ablation of cholinergic interneurons did not significantly change habitual responding or response substitution. Thus, activation -but not ablation -of cholinergic interneurons in the dorsolateral striatum modulates expression of a new habit when an old habit is replaced by a new one. Together with previous work, this suggests that striatal cholinergic interneurons facilitate behavioural flexibility in both dorsolateral striatum in addition to dorsomedial and ventral striatum.

Correction to: Cerebellar Modules and Their Role as Operational Cerebellar Processing Units: A Consensus paper.

In the original version of this paper, the Title should have been written with "A Consensus paper" to read "Cerebellar Modules and Their Role as Operational Cerebellar Processing Units: A Consensus paper".

Cerebellar Modules and Their Role as Operational Cerebellar Processing Units: A Consensus paper [corrected].

The compartmentalization of the cerebellum into modules is often used to discuss its function. What, exactly, can be considered a module, how do they operate, can they be subdivided and do they act individually or in concert are only some of the key questions discussed in this consensus paper. Experts studying cerebellar compartmentalization give their insights on the structure and function of cerebellar modules, with the aim of providing an up-to-date review of the extensive literature on this subject. Starting with an historical perspective indicating that the basis of the modular organization is formed by matching olivocorticonuclear connectivity, this is followed by consideration of anatomical and chemical modular boundaries, revealing a relation between anatomical, chemical, and physiological borders. In addition, the question is asked what the smallest operational unit of the cerebellum might be. Furthermore, it has become clear that chemical diversity of Purkinje cells also results in diversity of information processing between cerebellar modules. An additional important consideration is the relation between modular compartmentalization and the organization of the mossy fiber system, resulting in the concept of modular plasticity. Finally, examination of cerebellar output patterns suggesting cooperation between modules and recent work on modular aspects of emotional behavior are discussed. Despite the general consensus that the cerebellum has a modular organization, many questions remain. The authors hope that this joint review will inspire future cerebellar research so that we are better able to understand how this brain structure makes its vital contribution to behavior in its most general form.

Role of Striatal Cholinergic Interneurons in Set-Shifting in the Rat.

The ability to change strategies in different contexts is a form of behavioral flexibility that is crucial for adaptive behavior. The striatum has been shown to contribute to certain forms of behavioral flexibility such as reversal learning. Here we report on the contribution of striatal cholinergic interneurons-a key element in the striatal neuronal circuit-to strategy set-shifting in which an attentional shift from one stimulus dimension to another is required. We made lesions of rat cholinergic interneurons in dorsomedial or ventral striatum using a specific immunotoxin and investigated the effects on set-shifting paradigms and on reversal learning. In shifting to a set that required attention to a previously irrelevant cue, lesions of dorsomedial striatum significantly increased the number of perseverative errors. In this condition, the number of never-reinforced errors was significantly decreased in both types of lesions. When shifting to a set that required attention to a novel cue, rats with ventral striatum lesions made more perseverative errors. Neither lesion impaired learning of the initial response strategy nor a subsequent switch to a new strategy when response choice was indicated by a previously relevant cue. Reversal learning was not affected. These results suggest that in set-shifting the striatal cholinergic interneurons play a fundamental role, which is dissociable between dorsomedial and ventral striatum depending on behavioral context. We propose a common mechanism in which cholinergic interneurons inhibit neurons representing the old strategy and enhance plasticity underlying exploration of a new rule.

Lesion in the lateral cerebellum specifically produces overshooting of the toe trajectory in leading forelimb during obstacle avoidance in the rat.

During locomotion, stepping over an obstacle under visual guidance is crucial to continuous safe walking. Studies of the role of the central nervous system in stepping movements have focused on cerebral cortical areas such as the primary motor cortex and posterior parietal cortex. There is speculation that the lateral cerebellum, which has strong anatomical connections with the cerebral cortex, also plays a key role in stepping movements over an obstacle, although this function of the lateral cerebellum has not yet been elucidated. Here we investigated the role of the lateral cerebellum during obstacle avoidance locomotion in rats with a lateral cerebellar lesion. A unilateral lesion in the lateral cerebellum did not affect limb movements during overground locomotion. Importantly, however, the lesioned animals showed overshooting of the toe trajectory specific to the leading forelimb ipsilateral to the lesion when stepping over an obstacle, and the peak toe position, in which the toe is maximally raised during stepping, shifted away from the upper edge of the obstacle. Recordings of EMG activity from elbow flexor and extensor muscles suggested that the overshooting toe trajectory in the ipsilateral leading forelimb possibly resulted from sustained elbow flexion and delayed elbow extension following prolonged activity of the biceps brachii. These results suggest that the lateral cerebellum specifically contributes to generating appropriate toe trajectories in the ipsilateral leading forelimb and to controlling related muscle activities in stepping over an obstacle, especially when accurate control of the distal extremity is achieved under visual guidance.

Contributions of phase resetting and interlimb coordination to the adaptive control of hindlimb obstacle avoidance during locomotion in rats: a simulation study.

Obstacle avoidance during locomotion is essential for safe, smooth locomotion. Physiological studies regarding muscle synergy have shown that the combination of a small number of basic patterns produces the large part of muscle activities during locomotion and the addition of another pattern explains muscle activities for obstacle avoidance. Furthermore, central pattern generators in the spinal cord are thought to manage the timing to produce such basic patterns. In the present study, we investigated sensory-motor coordination for obstacle avoidance by the hindlimbs of the rat using a neuromusculoskeletal model. We constructed the musculoskeletal part of the model based on empirical anatomical data of the rat and the nervous system model based on the aforementioned physiological findings of central pattern generators and muscle synergy. To verify the dynamic simulation by the constructed model, we compared the simulation results with kinematic and electromyographic data measured during actual locomotion in rats. In addition, we incorporated sensory regulation models based on physiological evidence of phase resetting and interlimb coordination and examined their functional roles in stepping over an obstacle during locomotion. Our results show that the phase regulation based on interlimb coordination contributes to stepping over a higher obstacle and that based on phase resetting contributes to quick recovery after stepping over the obstacle. These results suggest the importance of sensory regulation in generating successful obstacle avoidance during locomotion.

Examining normative values using the Cambridge neuropsychological test automated battery and developmental traits of executive functions among elementary school-aged children in Japan.

The Cambridge Neuropsychological Test Automated Battery (CANTAB) is a computerized and child-friendly neuropsychological assessment battery that includes subtests aimed at evaluating some aspects of executive functions. Using the CANTAB, this study aims to establish normative values based on the aspects of executive functions among school-aged children in Japan. The participants included 234 children (135 boys and 99 girls aged 6-12 years) enrolled in regular classes, without any clinical records of developmental disorders or educational support. The participants were grouped according to age (6-7, 8-9, and 10-12 years). Four CANTAB subtests, including spatial working memory (SWM) to assess spatial working memory, Stockings of Cambridge (SOC) to evaluate planning, intra/extradimensional set shift (IED) to evaluate attentional set shifting and flexibility, and stop signal task (SST) to evaluate inhibition, were administered to each participant. The results showed that performance in all the CANTAB subtests administered changed with age. Among the subtests, compared with performances in the SOC and IED, those in the SWM and SST improved earlier, thereby indicating that spatial working memory and inhibition develop earlier than planning as well as attentional set shifting and flexibility. Additionally, in the SST subtest, girls made fewer errors than boys did in the 6-7 years group. This study presents normative data of four CANTAB subtests according to age and sex among school-aged children in Japan. We expect that the findings will be used to develop effective tools for the early detection of and support for children with executive dysfunction.

Aggregation-prone A53T mutant of α-synuclein exaggerates methamphetamine neurotoxicity in SH-SY5Y cells: Protective role of cellular cholesterol.

The aim of this study is to examine the effects of wild type as well as a mutant (A53T) form of α-synuclein (α-syn) on neuronal cells exposed to methamphetamine (METH). SH-SY5Y human dopaminergic neuronal cells stably expressing exogenously added wild type (WT) or A53T α-syn were established for this purpose. Among the three types of cells, parental, WT α-syn-overexpressing, and A53T α-syn overexpressing SH-SY5Y cells (hereafter referred to as SH-SY5Y, WT SH-SY5Y, and A53T SH-SY5Y, respectively), only A53T SH-SY5Y cells showed significant loss of cell viability after exposure to 2 mM METH for 24 h. Transcriptome analysis using DNA microarray showed that METH induced genes for cholesterol biosynthesis in all of these three cell lines, suggesting that METH upregulates cellular cholesterol biosynthesis independently from cellular α-syn levels. Visualization of the cellular localization of free cholesterol showed that METH induces an aberrant intracellular accumulation of free cholesterol in all three cell lines. In addition, we observed the aggregation of α-syn into cytoplasmic granules, which was more apparent with A53T α-syn than WT α-syn, in cells exposed to METH. Furthermore, the cell death observed in METH-treated A53T SH-SY5Y cells was exaggerated by the addition of 2-hydroxypropyl-ß-cyclodextrin (CD), a substance used to extract cholesterol from cells. These results suggest that the aggregation of A53T α-syn in METH-treated cells should be involved in cell death. The upregulation of cellular biosynthesis and cholesterol accumulation by METH should play a protective role against A53T α-syn neurotoxicity in METH-treated SH-SY5Y cells.

Abnormal cortical responses elicited by audiovisual movies in patients with autism spectrum disorder with atypical sensory behavior: A magnetoencephalographic study.

BACKGROUND: Atypical sensory behavior disrupts behavioral adaptation in children with autism spectrum disorder (ASD); however, neural correlates of sensory dysfunction using magnetoencephalography (MEG) remain unclear. METHOD: We used MEG to measure the cortical activation elicited by visual (uni)/audiovisual (multisensory) movies in 46 children (7-14 years) were included in final analysis: 13 boys with atypical audiovisual behavior in ASD (AAV+), 10 without this condition, and 23 age-matched typically developing boys. RESULTS: The AAV+ group demonstrated an increase in the cortical activation in the bilateral insula in response to unisensory movies and in the left occipital, right superior temporal sulcus (rSTS), and temporal regions to multisensory movies. These increased responses were correlated with severity of the sensory impairment. Increased theta-low gamma oscillations were observed in the rSTS in AAV+. CONCLUSION: The findings suggest that AAV is attributed to atypical neural networks centered on the rSTS.

Sibling Cases of Charcot-Marie-Tooth Disease Type 4H with a Homozygous FGD4 Mutation and Cauda Equina Thickening.

Charcot-Marie-Tooth disease type 4H (CMT4H) is an autosomal recessive inherited demyelinating neuropathy caused by an FYVE, RhoGEF, and a PH domain-containing protein 4 (FGD4) gene mutation. CMT4H is characterized by an early onset, slow progression, scoliosis, distal muscle atrophy, and foot deformities. We herein present sibling cases of CMT4H with a homozygous mutation in the FGD4 gene. Both patients exhibited cauda equina thickening on magnetic resonance imaging, which had not been reported among the previous CMT4H cases. This is the first report of CMT4H with a homozygous FGD4 c.1730G>A (p.Arg577Gln) mutation showing mild progression and cauda equina thickening.

Anti-LRP4 Antibody-associated Myasthenia Gravis with a Rare Complication of Thymoma Successfully Treated by Thymectomy.

We herein report the case of a 65-year-old woman diagnosed with myasthenia gravis (MG) after complaining of double vision. The patient had anti-low-density lipoprotein receptor-related protein 4 (LRP4) antibody in her serum, although antibodies against the acetylcholine receptor and muscle-specific tyrosine kinase were not detected. Chest computed tomography showed an anterior mediastinal tumor with a high uptake on fluorodeoxyglucose-positron emission tomography. Endoscopic thymectomy successfully ameliorated her ocular symptoms and showed the lesion to be thymoma. The present case revealed that anti-LRP4 antibody-associated MG can be associated with thymoma, which has been regarded as a rare complication of this disease thus far.

Lesser suppression of response to bright visual stimuli and visual abnormality in children with autism spectrum disorder: a magnetoencephalographic study.

BACKGROUND: Visual abnormality is a common sensory impairment in autism spectrum disorder (ASD), which may cause behavioral problems. However, only a few studies exist on the neural features corresponding to the visual symptoms in ASD. The purpose of this study was to investigate the relationship between cortical responses to visual stimuli and visual abnormality to examine the neurophysiological mechanisms of the visual abnormality in ASD. METHODS: Twenty-two high-functioning children with ASD (10.95 ± 2.01 years old) and 23 age-matched typically developing (TD) children (10.13 ± 2.80 years old) participated in this study. We measured the cortical responses (i.e., activated intensity and attenuation ratio) elicited by the Original visual image and other two types of bright images (the Dot noise or Blind image, which includes overlapped particles onto the Original image or the enhanced-brightness version of the Original image, respectively) using magnetoencephalography. RESULTS: The severity of visual abnormalities was significantly associated with behavioral problems in children with ASD. In addition, we found the increased cortical activation in response to the Original image in the left supramarginal gyrus (SMG) and middle temporal gyrus in children with ASD. However, there were no inter-group differences in the primary visual and medial orbitofrontal cortices. Furthermore, when we compared cortical responses according to the type of images, children with ASD showed lesser attenuation of the activated intensities than children with TD in response to the bright images compared with the Original image in the right SMG. These attenuation ratios (Dot noise/Original and Blind/Original) were also associated with the severity of visual abnormalities. CONCLUSIONS: Our results show that dysfunction of stimulus-driven neural suppression plays a crucial role in the neural mechanism of visual abnormality in children with ASD. To the best of our knowledge, this is the first magnetoencephalography study to demonstrate the association between the severity of visual abnormality and lower attenuation ratios in children with ASD. Our results contribute to the knowledge of the mechanisms underlying visual abnormality in children with ASD, and may therefore lead to more effective diagnosis and earlier intervention.

An open cortico-basal ganglia loop allows limbic control over motor output via the nigrothalamic pathway.

Cortico-basal ganglia-thalamocortical loops are largely conceived as parallel circuits that process limbic, associative, and sensorimotor information separately. Whether and how these functionally distinct loops interact remains unclear. Combining genetic and viral approaches, we systemically mapped the limbic and motor cortico-basal ganglia-thalamocortical loops in rodents. Despite largely closed loops within each functional domain, we discovered a unidirectional influence of the limbic over the motor loop via ventral striatum-substantia nigra (SNr)-motor thalamus circuitry. Slice electrophysiology verifies that the projection from ventral striatum functionally inhibits nigro-thalamic SNr neurons. In vivo optogenetic stimulation of ventral or dorsolateral striatum to SNr pathway modulates activity in medial prefrontal cortex (mPFC) and motor cortex (M1), respectively. However, whereas the dorsolateral striatum-SNr pathway exerts little impact on mPFC, activation of the ventral striatum-SNr pathway effectively alters M1 activity. These results demonstrate an open cortico-basal ganglia loop whereby limbic information could modulate motor output through ventral striatum control of M1.

Expression analysis of two SLAM family receptors, SLAMF2 and SLAMF7, in patients with multiple myeloma.

Monoclonal antibodies against surface antigens on MM cells, such as anti-SLAMF7 and anti-CD38 antibodies, represent an attractive therapeutic modality for the eradication of multiple myeloma (MM) cells. However, further exploration of target molecules is urgently needed for the development of more effective therapies. In the present study, we studied the expression of CD48 in a total of 74 primary MM samples derived from patients to evaluate SLAMF2 (CD48) as a candidate in mAb therapy for MM. Of 74 samples, 39 were subjected to SLAMF7 analysis. Most of the MM cells, defined as CD38 and CD138 double-positive cells, showed strong expression of CD48 or SLAMF7 independent of disease stage or treatment history. In these 39 samples, most MM cells showed expression of both SLAMF7 and CD48; however, several samples showed expression of either only CD48 or only SLAMF7, including seven cases that were only highly positive for SLAMF7, and five that were only highly positive for CD48. Our study demonstrates that the immune receptor CD48 is overexpressed on MM cells together with SLAMF7, and that CD48 may be considered as an alternative target for treatment of MM in cases showing weak expression of SLAMF7.

Pauses in cholinergic interneuron firing exert an inhibitory control on striatal output in vivo.

The cholinergic interneurons (CINs) of the striatum are crucial for normal motor and behavioral functions of the basal ganglia. Striatal CINs exhibit tonic firing punctuated by distinct pauses. Pauses occur in response to motivationally significant events, but their function is unknown. Here we investigated the effects of pauses in CIN firing on spiny projection neurons (SPNs) - the output neurons of the striatum - using in vivo whole cell and juxtacellular recordings in mice. We found that optogenetically-induced pauses in CIN firing inhibited subthreshold membrane potential activity and decreased firing of SPNs. During pauses, SPN membrane potential fluctuations became more hyperpolarized and UP state durations became shorter. In addition, short-term plasticity of corticostriatal inputs was decreased during pauses. Our results indicate that, in vivo, the net effect of the pause in CIN firing on SPNs activity is inhibition and provide a novel mechanism for cholinergic control of striatal output.

STAT3 is constitutively activated in chronic active Epstein-Barr virus infection and can be a therapeutic target.

Chronic active Epstein-Barr virus infection (CAEBV) is a lymphoproliferative disorder characterized by the clonal proliferation of EBV-infected T or NK cells and is related to severe systemic inflammation. This study aims to investigate STAT3 to elucidate the mechanism underlying the CAEBV development. We determined that STAT3 was constitutively activated in EBV-positive T- or NK-cell lines. We also determined that STAT3 was activated in the peripheral blood mononuclear cells (PBMCs) containing EBV-infected clonally proliferating T or NK cells in six of seven patients with CAEBV. We conducted direct sequencing of the STAT3 Src homology 2 (SH2) domain, which has previously been reported to be mutated in T- or NK-cell neoplasms. No mutation was detected in the STAT3 SH2 domain in patients with CAEBV. Next, we investigated the effects of ruxolitinib, an inhibitor of both JAK1 and JAK2, which phosphorylates and activates STAT3. Ruxolitinib suppressed the phosphorylation of STAT3 in EBV-positive T- or NK-cell lines. Ruxolitinib also decreased the viable cell number of EBV-positive T- or NK-cell lines and PBMCs from patients with CAEBV. Furthermore, ruxolitinib suppressed the production of inflammatory cytokines in the cell lines and CAEBV patient-derived cells. In conclusion, constitutively activated STAT3, which promotes survival and cytokine production, could be a therapeutic target for CAEBV.

Potent antitumor effect of combination therapy with sub-optimal doses of Akt inhibitors and pomalidomide plus dexamethasone in multiple myeloma.

Afuresertib (AFU), a novel inhibitor of the serine/threonine kinase AKT, has clinical efficacy as a monotherapy against hematological malignancies and is expected to be used in combination with standard therapies for multiple myeloma (MM). To develop a more effective and less toxic combination of immunomodulatory drugs (IMiDs) for therapy, the antitumor effect of sub-optimal doses of AFU, pomalidomide plus dexamethasone (PD), and the AFU-PD combination on MM cells were examined in the present study. Two MM cell lines, XG-7 and U266, with low sensitivity to both PD and AFU monotherapies, were subjected to these combinations and analyzed. Although the cell lines showed a slight reduction in viability with the sub-optimal doses of each monotherapy, the combination of the treatments resulted in a reduction in cell viability and the progression of apoptosis. Co-treatment with sub-optimal doses of PD and AFU enhanced caspase activation and highly suppressed the expression of IKZF1 and IKZF3. In addition, this combination promoted the dephosphorylation and stabilization of 4EBP1, an inhibitor of eIF4E activation, which led to the impairment of eIF4E-mediated translational activity. Furthermore, AFU showed a sufficient inhibitory effect on the phosphorylation of FOXO1, a tumor suppressor, in monotherapy or in combination with PD, which may be attributable to the activation of FOXO1, the subsequent inhibition of tumor growth, and the induction of cell death. In conclusion, the combination therapy with sub-optimal doses of PD and AFU exhibited potent antitumor activity in MM cells and may provide a novel strategy for the treatment of patients who experienced intolerable toxicity or insufficient response during IMiD therapy.