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Tissue-clearing techniques are powerful tools for biological research and pathological diagnosis. Here, we describe advanced clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) procedures that can be applied to biomedical research. This protocol enables preparation of high-transparency organs that retain fluorescent protein signals within 7-21 d by immersion in CUBIC reagents. A transparent mouse organ can then be imaged by a high-speed imaging system (0.5 TB/h/color). In addition, to improve the understanding and simplify handling of the data, the positions of all detected cells in an organ (3-12 GB) can be extracted from a large image dataset (2.5-14 TB) within 3-12 h. As an example of how the protocol can be used, we counted the number of cells in an adult whole mouse brain and other distinct anatomical regions and determined the number of cells transduced with mCherry following whole-brain infection with adeno-associated virus (AAV)-PHP.eB. The improved throughput offered by this protocol allows analysis of numerous samples (e.g.,100 mouse brains per study), providing a platform for next-generation biomedical research.

Restoration of damaged central nervous system structures, functional recovery, and prevention of neuronal loss during neurodegenerative diseases are major objectives in cerebellar research. The highly organized anatomical structure of the cerebellum with numerous inputs/outputs, the complexity of cerebellar functions, and the large spectrum of cerebellar ataxias render therapies of cerebellar disorders highly challenging. There are currently several therapeutic approaches including motor rehabilitation, neuroprotective drugs, non-invasive cerebellar stimulation, molecularly based therapy targeting pathogenesis of the disease, and neurotransplantation. We discuss the goals and possible beneficial mechanisms of transplantation therapy for cerebellar damage and its limitations and factors determining outcome.

The c-RET proto-oncogene encodes a receptor-tyrosine kinase. Loss-of-function mutations of RET have been shown to be associated with Hirschsprung disease and Down's syndrome (HSCR-DS) in humans. DS is known to involve cerebellar hypoplasia, which is characterized by reduced cerebellar size. Despite the fact that c-Ret has been shown to be associated with HSCR-DS in humans and to be expressed in Purkinje cells (PCs) in experimental animals, there is limited information about the role of activity of c-Ret/c-RET kinase in cerebellar hypoplasia. We found that a loss-of-function mutation of c-Ret Y1062 in PCs causes cerebellar hypoplasia in c-Ret mutant mice. Wild-type mice had increased phosphorylation of c-Ret in PCs during postnatal development, while c-Ret mutant mice had postnatal hypoplasia of the cerebellum with immature neurite outgrowth in PCs and granule cells (GCs). c-Ret mutant mice also showed decreased numbers of glial fibers and mitogenic sonic hedgehog (Shh)-positive vesicles in the external germinal layer of PCs. c-Ret-mediated cerebellar hypoplasia was rescued by subcutaneous injection of a smoothened agonist (SAG) as well as by reduced expression of Patched1, a negative regulator for Shh. Our results suggest that the loss-of-function mutation of c-Ret Y1062 results in the development of cerebellar hypoplasia via impairment of the Shh-mediated development of GCs and glial fibers in mice with HSCR-DS.

Background Adeno-associated virus (AAV) vectors have excellent properties as gene transfer vehicles. The recent development of AAV-PHP.eB, highly BBB-permeable capsid variant of AAV serotype 9, has opened up systemic application for whole brain transduction. To attain high transduction efficacy, much efforts have been paid to purify AAV vectors using gradient centrifugation or column chromatography. These methods are time-consuming, cost substantially and require expensive equipment. New method We propose a simple purification method for the production of systemically applicable AAV-PHP.eB targeting the brain. The new method, which we named m inimal p urification (MP) method, requires only 2 steps: removal of cell debris using a syringe filter and concentration using a disposable ultrafiltration device. Results The MP method yielded 2 times more AAV-PHP.eB than the standard ultracentrifuge purification (UCP) method. Intravenous injection of AAV-PHP.eB prepared using the MP method caused robust whole brain transduction without overt toxicity on the liver and kidney. Moreover, we found almost no difference in cellular density and morphology of brain microglia between control mice and mice treated systemically with the MP viral solution, suggesting no influence of the viral injection on brain immunity. Comparison with existing methods The new method, which requires only a benchtop centrifuge and takes only 2–4 h to obtain a ready-to-use viral solution, is much less expensive than the existing UCP method, and can avoid cumbersome and time-consuming purification processes. Conclusions This simplified method further expands the use of AAV vectors in the neuroscience community.

Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disease that presents with cerebellar ataxia and motor learning defects. Previous studies have indicated that the pathology of SCA1, as well as other ataxic diseases, is related to signalling pathways mediated by the metabotropic glutamate receptor type 1 (mGluR1), which is indispensable for proper motor coordination and learning. However, the functional contribution of mGluR signalling to SCA1 pathology is unclear. In the present study, we show that SCA1 model mice develop a functional impairment of mGluR signalling which mediates slow synaptic responses, dendritic Ca2+ signals and short- and long-term synaptic plasticity at parallel fibre (PF)-Purkinje cell (PC) synapses in a progressive manner from the early disease stage (5 postnatal weeks) prior to PC death. Notably, impairment of mGluR-mediated dendritic Ca2+ signals linearly correlated with reduction of PC capacitance (cell surface area) in the disease progression. Enhancement of mGluR signalling by baclofen, a clinically available GABAB receptor agonist, led to an improvement of motor performance in SCA1 mice and the improvement lasted ∼1 week after a single application of baclofen. Moreover, the restoration of motor performance in baclofen-treated SCA1 mice matched the functional recovery of mGluR-mediated slow synaptic currents and mGluR-dependent short- and long-term synaptic plasticity. These results suggest that impairment of synaptic mGluR cascades is one of the important contributing factors to cerebellar ataxia in early and middle stages of SCA1 pathology, and that modulation of mGluR signalling by baclofen or other clinical interventions may be therapeutic targets to treat SCA1. This article is protected by copyright. All rights reserved

Summary Aims Spinocerebellar ataxia type 1 (SCA1) is caused by the ataxin-1 protein (ATXN1) with an abnormally expanded polyglutamine tract and is characterized by progressive neurodegeneration. We previously showed that intrathecal injection of mesenchymal stem cells (MSCs) during the nonsymptomatic stage mitigates the degeneration of the peripheral nervous system (PNS) neurons in SCA1-knock-in (SCA1-KI) mice. We tested in this study whether the therapeutic effects of MSCs in SCA1-KI mice could be reproduced with MSC-releasing factor(s). Methods To test the effects of MSC-releasing factor(s), we used MSC-conditioned medium (MSC-CM). MSC-CM was intrathecally and/or intravenously injected into young SCA1-KI mice, and the therapeutic effects were assessed in the PNS at later ages using immunostaining, electrophysiology, and behavioral tests. Results MSC-CM attenuated the degeneration of axons and myelin of spinal motor neurons. Consequently, the injected SCA1-KI mice exhibited smaller reductions in nerve conduction velocity in spinal motor neurons and reduced motor incoordination than the untreated mice. Conclusions These results suggest that factors released from MSC mitigate the morphological and functional abnormalities in the PNS that are observed in SCA1-KI mice in a paracrine manner.

Others and we showed that RNA interference holds great promise for the treatment of dominantly inherited neurodegenerative disorders such as Machado-Joseph disease (MJD), for which there is no available treatment. However, successful experiments involved intracranial administration of viral vectors and there is a need for a safer and less invasive procedure. In this work, we successfully generated stable nucleic acid lipid particles (SNALPs), incorporating a short peptide derived from rabies virus glycoprotein (RVG-9r) and encapsulating small interfering RNAs (siRNAs), which can target mutant ataxin-3. The developed formulation exhibited important features that make it adequate for systemic administration: high encapsulation efficiency of siRNAs, ability to protect the encapsulated siRNAs, appropriate and homogeneous particle size distribution. Following optimization of the formulation and in vitro validation of its efficacy to silence the MJD-causing protein - mutant ataxin-3 - in neuronal cells, in vivo experiments showed that intravenous administration of RVG-9r-targeted SNALPs efficiently silenced mutant ataxin-3 reducing neuropathology and motor behavior deficits in two mouse models of MJD. To our knowledge, this is the first report showing beneficial impact of a non-viral gene silencing strategy in MJD and the first time that a non-invasive systemic administration proved to be beneficial on a polyglutamine disorder. Our study opens new avenues towards MJD therapy that can also be applied to other neurodegenerative diseases linked to the production of pathogenic proteins.

In a clinical setting, where multiple administrations of the therapeutic agent are usually required to improve the therapeutic outcome, it is crucial to assess the immunogenicity of the administered nanoparticles. In this data work, we investigated the safety profile of the repeated intravenous administration of brain-targeted stable nucleic acid lipid particles (RVG-9r-targeted SNALPs). To evaluate local activation of the immune system, we performed analysis of mouse tissue homogenates and sections from cerebellum. To investigate peripheral activation of the immune system, we used serum of mice that were intravenously injected with RVG-9r-targeted SNALPs. These data are related and were discussed in the accompanying research article entitled “Intravenous administration of brain-targeted stable nucleic acid lipid particles alleviates Machado–Joseph disease neurological phenotype” (Conceição et al., in press) [1].

Transcranical direct current stimulation (tDCS) is a treatment known to ameliorate various neurological conditions and enhance memory and cognition in humans. tDCS has gained traction for its potential therapeutic value; however, little is known about its mechanism of action. Using a transgenic mouse expressing G-CaMP7 in astrocytes and a subpopulation of excitatory neurons, we find that tDCS induces large-amplitude astrocytic Ca2+ surges across the entire cortex with no obvious changes in the local field potential. Moreover, sensory evoked cortical responses are enhanced after tDCS. These enhancements are dependent on the alpha-1 adrenergic receptor and are not observed in IP3R2 (inositol trisphosphate receptor type 2) knockout mice, in which astrocytic Ca2+ surges are absent. Together, we propose that tDCS changes the metaplasticity of the cortex through astrocytic Ca2+/IP3 signalling., While transcranical direct current stimulation (tDCS) is used in clinical setting, its cellular mechanism of action is unclear. Here, Hajime Hirase and colleagues visualize cellular response in mouse brain to tDCS and show robust astrocyte activation that coincide with plasticity changes.

Spinocerebellar ataxia type 1 (SCA1) is a devastating neurodegenerative disorder in which an abnormally expanded polyglutamine tract is inserted into causative ataxin-1 proteins. We have previously shown that SCA1 knockin (SCA1-KI) mice over 6 months of age exhibit a degeneration of motor neuron axons and their encasing myelin sheaths, as reported in SCA1 patients. We examined whether axon degeneration precedes myelin degeneration or vice versa in SCA1-KI mice and then attempted to mitigate motor neuron degeneration by intrathecally administering mesenchymal stem cells (MSCs). Temporal examination of the diameters of motor neuron axons and their myelin sheaths revealed a decrease in diameter of the axon but not of the myelin sheaths in SCA1-KI mice as early as 1 month of age, which suggests secondary degeneration of the myelin sheaths. We injected MSCs into the intrathecal space of SCA1-KI mice at 1 month of age, which resulted in a significant suppression of degeneration of both motor neuron axons and myelin sheaths, even 6 months after the MSC injection. Thus, MSCs effectively suppressed peripheral nervous system degeneration in SCA1-KI mice. It has not yet been clarified how clinically administered MSCs exhibit significant therapeutic effects in patients with SCA1. The morphological evidence presented in this current mouse study might explain the mechanisms that underlie the therapeutic effects of MSCs that are observed in patients with SCA1. © 2015 Wiley Periodicals, Inc.

Motor learning involves neural circuit modifications in the cerebellar cortex, likely through re-weighting of parallel fiber inputs onto Purkinje cells (PCs). Climbing fibers instruct these synaptic modifications when they excite PCs in conjunction with parallel fiber activity, a pairing that enhances climbing fiber-evoked Ca2+ signaling in PC dendrites. In vivo, climbing fibers spike continuously, including during movements when parallel fibers are simultaneously conveying sensorimotor information to PCs. Whether parallel fiber activity enhances climbing fiber Ca2+ signaling during motor behaviors is unknown. In mice, we found that inhibitory molecular layer interneurons (MLIs), activated by parallel fibers during practiced movements, suppressed parallel fiber enhancement of climbing fiber Ca2+ signaling in PCs. Similar results were obtained in acute slices for brief parallel fiber stimuli. Interestingly, more prolonged parallel fiber excitation revealed latent supralinear Ca2+ signaling. Therefore, the balance of parallel fiber and MLI input onto PCs regulates concomitant climbing fiber Ca2+ signaling.

Hydrogen sulfide and reactive sulfur species are regulators of physiological functions, have antioxidant effects against oxidative stresses, and are endogenously generated from l-cysteine. Recently, a novel pathway that generates hydrogen sulfide and reactive sulfur species from d-cysteine has been identified. d-Amino acid oxidase (DAO) is involved in this pathway and, among the various brain regions, is especially abundant in the cerebellum. d-Cysteine has been found to be a better substrate in the generation of hydrogen sulfide in the cerebellum than l-cysteine. Therefore, d-cysteine might be a novel neuroprotectant against cerebellar diseases such as spinocerebellar ataxia (SCA). However, it remains unknown if d-cysteine affects cerebellar Purkinje cells (PCs), which are important for cerebellar functions and are frequently degenerated in SCA patients. In the present study, we investigated whether the production of hydrogen sulfide from d-cysteine affects the dendritic development of cultured PCs. d-Cysteine was found to enhance the dendritic development of PCs significantly, while l-cysteine impaired it. The effect of d-cysteine was inhibited by simultaneous treatment with DAO inhibitors and was reproduced by treatment with 3-mercaptopyruvate, a metabolite of d-cysteine produced by the action of DAO, and disodium sulfide, a donor of hydrogen sulfide. In addition, hydrogen sulfide was immediately produced in cerebellar primary cultures after treatment with d-cysteine and 3-mercaptopyruvate. These findings suggest that d-cysteine enhances the dendritic development of primary cultured PCs via the generation of hydrogen sulfide.

We previously developed an industrial production process for novel water-soluble indigestible polysaccharides (resistant glucan mixture, RGM). During the process, an anhydrosugar—levoglucosan —is formed as a by-product and needs to be removed to manufacture a complete non-calorie product. Here, we attempted to isolate thermophilic bacteria that utilize levoglucosan as a sole carbon source, to establish a removing process for levoglucosan at higher temperature. Approximately 800 natural samples were used to isolate levoglucosan-utilizing microorganisms. Interestingly, levoglucosan-utilizing microorganisms—most of which were filamentous fungi or yeasts—could be isolated from almost all samples at 25°C. We isolated three thermophilic bacteria that grew well on levoglucosan medium at 60°C. Two of them and the other were identified as Bacillus smithii and Parageobacillus thermoglucosidasius, respectively, by 16S rDNA sequence analysis. Using B. smithii S-2701M, which showed best growth on levoglucosan, glucose and levoglucosan in 5% (wt/vol) RGM were completely diminished at 50°C for 144 h. These bacteria are known to have a biotechnological potential, given that they can ferment a range of carbon sources. This is the first report in the utilization of levoglucosan by these thermophiles, suggesting that our results expand their biotechnological potential for the unutilized carbon resources.

Adeno-associated virus (AAV)-PHP.B, a capsid variant of AAV serotype 9, is highly permeable to the blood-brain barrier. A major obstacle to the systemic use of AAV-PHP.B is the generation of neutralizing antibodies (NAbs); however, temporal profiles of NAb production after exposure to AAV-PHP.B, and the influence on later AAV-PHP.B administration, remains unknown. To address these, AAV-PHP.Bs expressing either GFP or mCherry by neuron-specific or astrocyte-specific promoters were intravenously administered to mice at various intervals, and brain expression was examined. Injection of two AAV-PHP.Bs, separated temporally, showed that as little as a 1-day interval between injections resulted in a significant decrease in expression of the second transgene, with a complete loss of expression after 7 days, paralleling an increase in serum NAb titers. Brain parenchymal injection was explored to circumvent the presence of NAbs. Mice systemically pre-treated with an AAV-PHP.B were injected intra-cerebrally with an AAV-PHP.B expressing GFP. After 2 weeks, marked GFP expression in the cerebellum was evident, showing that pre-existing NAbs did not affect the AAV-PHP.B directly injected into the brain. In contrast, reversing the injection order, i.e., cerebellar injection followed by systemic injection, completely eliminated expression of the second transgene. We confirmed that intra-cerebellar injection produced NAbs in the serum, but not in the cerebrospinal fluid (CSF). Our results indicate that the preclusion of brain transduction by a second AAV-PHP.B administration begins from the first day following systemic injection and is established within 1 week. Serum NAbs can be avoided by directly injecting AAV-PHP.Bs into brain tissue.

Neuronal Elav-like (nElavl or neuronal Hu) proteins are RNA-binding proteins that regulate RNA stability and alternative splicing, which are associated with axonal and synaptic structures. nElavl proteins promote the differentiation and maturation of neurons via their regulation of RNA. The functions of nElavl in mature neurons are not fully understood, although Elavl3 is highly expressed in the adult brain. Furthermore, possible associations between nElavl genes and several neurodegenerative diseases have been reported. We investigated the relationship between nElavl functions and neuronal degeneration using Elavl3−/− mice. Elavl3−/− mice exhibited slowly progressive motor deficits leading to severe cerebellar ataxia, and axons of Elavl3−/− Purkinje cells were swollen (spheroid formation), followed by the disruption of synaptic formation of axonal terminals. Deficit in axonal transport and abnormalities in neuronal polarity was observed in Elavl3−/− Purkinje cells. These results suggest that nElavl proteins are crucial for the maintenance of axonal homeostasis in mature neurons. Moreover, Elavl3−/− mice are unique animal models that constantly develop slowly progressive axonal degeneration. Therefore, studies of Elavl3−/− mice will provide new insight regarding axonal degenerative processes.

Protein kinase C (PKC), a family of serine/threonine protein kinases, mediates a myriad of patho-physiological cellular events in various tissues. The originally discovered PKC (conventional) requires the binding of diacylglycerol and Ca2+ for full activation. The conventional PKC consists of four isoforms, PKCα, PKCβI/βII, and PKCγ. PKCα and PKCβI/βII are expressed in the cells of various tissues including the brain, while PKCγ is present specifically in neurons of the brain and spinal cord. The cerebellum expresses the largest amount of PKC with all its four isoforms. Purkinje cells express PKCα and PKCγ. Previous studies have shown that PKCα is involved in the induction of long-term depression (LTD) at parallel fiber-Purkinje cell synapses. On the other hand, analysis of PKCγ-deficient mice has revealed that PKCγ plays a critical role in eliminating supernumerary climbing fiber synapses from developing Purkinje cells. Although why PKCα has no compensatory action in climbing fiber pruning in PKCγ-deficient Purkinje cells had so far remained unclear, we have recently demonstrated that PKCα is also capable of pruning supernumerary climbing fiber synapses, but the expression levels of PKCα are too low to achieve pruning in PKCγ-null Purkinje cells. Notably, although PKCγ is most abundant in Purkinje cells, its physiological role in mature Purkinje cells remained totally unknown. In addition to a concise review of the physiological and pathological roles of conventional PKCs in Purkinje cells, this report postulates a contribution of PKCα in developing Purkinje cells and a possible involvement of PKCγ in motor coordination in the mature cerebellum.

Retinoid-related orphan receptor α (RORα) is a critical master transcription factor that governs postnatal cerebellar development. An RORα-deficient cerebellum has a persistent external granular layer (EGL), rudimentary Purkinje cell (PC) dendrites, grossly reduced numbers of immature parallel fiber (PF)-PC synapses, and multiple climbing fibers (CF) innervating PCs in mice after 3 weeks of age when these features have disappeared in wild-type mice. Functionally, metabotropic glutamate receptor (mGluR)-mediated signaling in PCs is completely abrogated. Here we examined whether these defects could be corrected by lentivirally providing the RORα gene to 3-week-old PCs of RORα-deficient homozygous staggerer (sg/sg) mice. RORα expression in sg/sg PCs significantly increased the numbers of PF–PC synapses, spines on PC dendritic branchlets, and internal granule cells, concomitant with regression of the EGL, suggesting enhanced proliferation in the EGL and migration of post-mitotic progeny into the internal granular layer with augmented synaptogenesis between PFs and PC dendrites. However, the primary dendritic stems were only slightly extended, and mGluR signaling and the loss of redundant CF synapses in sg/sg PCs remained unrestored. These results suggest that the mitogenic and migratory potential of external granule cells in response to RORα was preserved in the >3-week-old sg/sg mouse cerebellum. Moreover, sg/sg PCs sprouted spines and formed synapses with PFs. However, lengthening of the primary dendritic stems, establishment of mGluR signaling, and removal of CF synapses in sg/sg PCs were regressed by 3 weeks of age.

Shp2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) regulates neural cell differentiation. It is also expressed in postmitotic neurons, however, and mutations of Shp2 are associated with clinical syndromes characterized by mental retardation. Here we show that conditional-knockout (cKO) mice lacking Shp2 specifically in postmitotic forebrain neurons manifest abnormal behavior, including hyperactivity. Novelty-induced expression of immediate-early genes and activation of extracellular-signal-regulated kinase (Erk) were attenuated in the cerebral cortex and hippocampus of Shp2 cKO mice, suggestive of reduced neuronal activity. In contrast, ablation of Shp2 enhanced high-K(+)-induced Erk activation in both cultured cortical neurons and synaptosomes, whereas it inhibited that induced by brain-derived growth factor in cultured neurons. Posttetanic potentiation and paired-pulse facilitation were attenuated and enhanced, respectively, in hippocampal slices from Shp2 cKO mice. The mutant mice also manifested transient impairment of memory formation in the Morris water maze. Our data suggest that Shp2 contributes to regulation of Erk activation and synaptic plasticity in postmitotic forebrain neurons and thereby controls locomotor activity and memory formation.

Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disease caused by extension of a CAG repeat in the Sca1gene. Although the mechanisms underlying the symptoms of SCA1 have not been determined, aberrant neuronal activation potentially contributes to the neuronal cell death characteristic of the disease. Here we examined the potential involvement of extrasynaptic N-methyl-d-aspartate receptor (NMDAR) activation in the pathogenesis of SCA1 by administering memantine, a low-affinity noncompetitive NMDAR antagonist, in SCA1 knock-in (KI) mice. In KI mice, the exon in the ataxin 1 gene is replaced with abnormally expanded 154CAG repeats. Memantine was administered orally to the SCA1 KI mice from 4 weeks of age until death. The treatment significantly attenuated body-weight loss and prolonged the life span of SCA1 KI mice. Furthermore, memantine significantly suppressed the loss of Purkinje cells in the cerebellum and motor neurons in the dorsal motor nucleus of the vagus, which are critical for motor function and parasympathetic function, respectively. These findings support the contribution of aberrant activation of extrasynaptic NMDARs to neuronal cell death in SCA1 KI mice and suggest that memantine may also have therapeutic benefits in human SCA1 patients.

Machado-Joseph disease is a neurodegenerative disease without effective treatment. Patients with Machado-Joseph disease exhibit significant motor impairments such as gait ataxia, associated with multiple neuropathological changes including mutant ATXN3 inclusions, marked neuronal loss and atrophy of the cerebellum. Thus, an effective treatment of symptomatic patients with Machado-Joseph disease may require cell replacement, which we investigated in this study. For this purpose, we injected cerebellar neural stem cells into the cerebellum of adult Machado-Joseph disease transgenic mice and assessed the effect on the neuropathology, neuroinflammation mediators and neurotrophic factor levels and motor coordination. We found that upon transplantation into the cerebellum of adult Machado-Joseph disease mice, cerebellar neural stem cells differentiate into neurons, astrocytes and oligodendrocytes. Importantly, cerebellar neural stem cell transplantation mediated a significant and robust alleviation of the motor behaviour impairments, which correlated with preservation from Machado-Joseph disease-associated neuropathology, namely reduction of Purkinje cell loss, reduction of cellular layer shrinkage and mutant ATXN3 aggregates. Additionally, a significant reduction of neuroinflammation and an increase of neurotrophic factors levels was observed, indicating that transplantation of cerebellar neural stem cells also triggers important neuroprotective effects. Thus, cerebellar neural stem cells have the potential to be used as a cell replacement and neuroprotective approach for Machado-Joseph disease therapy.

Neurodegenerative diseases such as spinocerebellar ataxias and autoantibody-associated disorders of the central nervous system often affect the cerebellum, resulting in motor deficits. Recent studies have revealed that most of these disorders impair type 1 metabotropic glutamate receptor (mGluR1) and/or the closely associated signaling molecules in cerebellar Purkinje cell. Since the signaling pathway triggered by mGluR1 activation in Purkinje cell plays a pivotal role in coordinated movements and motor learning, pharmacological repair of aberrant mGluR1 signaling in Purkinje cell is critical for mitigation of cerebellar symptoms. Here we review recently identified pathophysiology underlying the neurodegenerative and autoimmune diseases affecting mGluR1 signaling in Purkinje cell and possible therapeutic interventions.

cAMP is a common second messenger that is involved in various physiological processes. To expand the colour palette of available cAMP indicators, we developed a red cAMP indicator named “Pink Flamindo” (Pink Fluorescent cAMP indicator). The fluorescence intensity of Pink Flamindo increases 4.2-fold in the presence of a saturating dose of cAMP, with excitation and emission peaks at 567 nm and 590 nm, respectively. Live-cell imaging revealed that Pink Flamindo is effective for monitoring the spatio-temporal dynamics of intracellular cAMP generated by photoactivated adenylyl cyclase in response to blue light, and in dual-colour imaging studies using a green Ca2+ indicator (G-GECO). Furthermore, we successfully monitored the elevation of cAMP levels in vivo in cerebral cortical astrocytes by two-photon imaging. We propose that Pink Flamindo will facilitate future in vivo, optogenetic studies of cell signalling and cAMP dynamics.

Machado-Joseph disease or spinocerebellar ataxia type 3, the most common dominantly-inherited spinocerebellar ataxia, results from translation of the polyglutamine-expanded and aggregation prone ataxin 3 protein. Clinical manifestations include cerebellar ataxia and pyramidal signs and there is no therapy to delay disease progression. Beclin 1, an autophagy-related protein and essential gene for cell survival, is decreased in several neurodegenerative disorders. This study aimed at evaluating if lentiviral-mediated beclin 1 overexpression would rescue motor and neuropathological impairments when administered to pre- and post-symptomatic lentiviral-based and transgenic mouse models of Machado-Joseph disease. Beclin 1-mediated significant improvements in motor coordination, balance and gait with beclin 1-treated mice equilibrating longer periods in the Rotarod and presenting longer and narrower footprints. Furthermore, in agreement with the improvements observed in motor function beclin 1 overexpression prevented neuronal dysfunction and neurodegeneration, decreasing formation of polyglutamine-expanded aggregates, preserving Purkinje cell arborization and immunoreactivity for neuronal markers. These data show that overexpression of beclin 1 in the mouse cerebellum is able to rescue and hinder the progression of motor deficits when administered to pre- and post-symptomatic stages of the disease. * Abbreviation : SCA : spinocerebellar ataxia

Cerebellar Purkinje cells (PCs) express two members of the classical protein kinase C (cPKC) subfamily, namely, PKCα and PKCγ. Previous studies on PKCγ knockout (KO) mice have revealed a critical role of PKCγ in the pruning of climbing fibers (CFs) from PCs during development. The question remains as to why only PKCγ and not PKCα is involved in CF synapse elimination from PCs. To address this question, we assessed the expression levels of PKCγ and PKCα in wild-type (WT) and PKCγ KO PCs using PC-specific quantitative real-time reverse transcription-polymerase chain reaction, western blotting, and immunohistochemical analysis. The results revealed that the vast majority of cPKCs in PCs were PKCγ, whereas PKCα accounted for the remaining minimal fraction. The amount of PKCα was not up-regulated in PKCγ KO PCs. Lentiviral expression of PKCα in PKCγ KO PCs resulted in a 10-times increase in the amount of PKCα mRNA in the PKCγ KO PCs, compared to that in WT PCs. Our quantification showed that the expression levels of cPKC mRNA in PKCγ KO PCs increased roughly from 1% to 22% of that in WT PCs solely through PKCα expression. The up-regulation of PKCα in PKCγ KO PCs significantly rescued the impaired CF synapse elimination. Although both PKCα and PKCγ are capable of pruning supernumerary CF synapses from developing PCs, these results suggest that the expression levels of cPKCs in PKCγ KO PCs are too low for CF pruning.

We examined integrase-defective lentiviral vectors (IDLVs) with a mutant (D64V) integrase in terms of their residual integration capability, the levels and duration of transgene expression and their therapeutic potential in comparison to wild-type lentiviral vectors (WTLVs) with a wild-type integrase gene. Compared with WTLVs, the IDLV-mediated proviral integration into host-cell chromosomes was approximately 1/3850 in HeLa cells and approximately 1/111 in mouse cerebellar neurons in vivo. At 2 months, transgene expression by IDLVs in the mouse cerebellum was comparable to that by WTLVs, but then significantly decreased. The mRNA levels at 6 and 12 months after injection in IDLV-infected cerebella were approximately 26% and 5%, respectively, of the mRNA levels in WTLV-injected cerebella. To examine the therapeutic potential, IDLVs or WTLVs expressing a molecule that enhances the ubiquitin-proteasome pathway were injected into the cerebella of spinocerebellar ataxia type 3 model mice (SCA3 mice). IDLV-injected SCA3 mice showed a significantly improved rotarod performance even at 1 year after-injection. Immunohistochemistry at 1 year after injection showed a drastic reduction of mutant aggregates in Purkinje cellsfrom IDLV-injected, as well as WTLV-injected, SCA3 mice. Our results suggest that because of the substantially reduced risk of insertional mutagenesis, IDLVs are safer and potentially effective as gene therapy vectors.

Neural circuits are shaped by elimination of early-formed redundant synapses during postnatal development. Retrograde signaling from postsynaptic cells regulates synapse elimination. In this work, we identified semaphorins, a family of versatile cell recognition molecules, as retrograde signals for elimination of redundant climbing fiber to Purkinje cell synapses in developing mouse cerebellum. Knockdown of Sema3A, a secreted semaphorin, in Purkinje cells or its receptor in climbing fibers accelerated synapse elimination during postnatal day 8 (P8) to P18. Conversely, knockdown of Sema7A, a membrane-anchored semaphorin, in Purkinje cells or either of its two receptors in climbing fibers impaired synapse elimination after P15. The effect of Sema7A involves signaling by metabotropic glutamate receptor 1, a canonical pathway for climbing fiber synapse elimination. These findings define how semaphorins retrogradely regulate multiple processes of synapse elimination.

Postnatal hematopoietic progenitor cells do not contribute to microglial homeostasis in adult mice under normal conditions. However, previous studies using whole-body irradiation and bone marrow (BM) transplantation models have shown that adult BM cells migrate into the brain tissue and differentiate into microglia (BM-derived microglia; BMDM). Here, we investigated whether cranial irradiation alone was sufficient to induce the generation of BMDM in the adult mouse brain. Transgenic mice that express green fluorescent protein (GFP) under the control of a murine stem cell virus (MSCV) promoter (MSCV-GFP mice) were used. MSCV-GFP mice express GFP in BM cells but not in the resident microglia in the brain. Therefore, these mice allowed us to detect BM-derived cells in the brain without BM reconstitution. MSCV–GFP mice, aged 8–12 weeks, received 13.0 Gy irradiation only to the cranium, and BM-derived cells in the brain were quantified at 3 and 8 weeks after irradiation. No BM-derived cells were detected in control non-irradiated MSCV-GFP mouse brains, but numerous GFP-labeled BM-derived cells were present in the brain stem, basal ganglia and cerebral cortex of the irradiated MSCV-GFP mice. These BM-derived cells were positive for Iba1, a marker for microglia, indicating that GFP-positive BM-derived cells were microglial in nature. The population of BMDM was significantly greater at 8 weeks post-irradiation than at 3 weeks post-irradiation in all brain regions examined. Our results clearly show that cranial irradiation alone is sufficient to induce the generation of BMDM in the adult mouse.

Background Certain inherited progressive neurodegenerative disorders, such as spinocerebellar ataxia (SCA), affect neurons in large areas of the central nervous system (CNS). The selective expression of disease-causing and therapeutic genes in susceptible regions and cell types is critical for the generation of animal models and development of gene therapies for these diseases. Previous studies have demonstrated the advantages of the short synapsin I (SynI) promoter (0.5 kb) as a neuron-specific promoter for robust transgene expression. However, the short SynI promoter has also shown some promoter activity in glia and a lack of transgene expression in significant areas of the CNS. New methods: To improve the SynI promoter, we used a SynI promoter that is twice as long (1.0 kb) as the short SynI promoter and incorporated a minimal CMV (minCMV) sequence. Results We observed that the 1.0 kb rat SynI promoter with minCMV [rSynI(1.0)-minCMV] exhibited robust promoter strength, excellent neuronal specificity and wide-ranging transgene expression throughout the CNS. Comparison with existing methods: Compared with the two previously reported short (0.5 kb) promoters, the new promoter was superior with respect to neuronal specificity and more efficiently transduced neurons. Moreover, transgenic mice expressing the mutant protein ATXN1[Q98], which causes SCA type 1 (SCA1), under the control of the rSynI(1.0)-minCMV promoter showed robust transgene expression specifically in neurons throughout the CNS and exhibited progressive ataxia. Conclusion rSynI(1.0)-minCMV drives robust and neuron-specific transgene expression throughout the CNS and is therefore useful for viral vector-mediated neuron-specific gene delivery and generation of neuron-specific transgenic animals.

Glial development is critical for the function of the central nervous system. CD38 is a multifunctional molecule with ADP-ribosyl cyclase activity. While critical roles of CD38 in the adult brain such as oxytocin release and social behavior have been reported, those in the developing brain remain largely unknown. Here we demonstrate that deletion of Cd38 leads to impaired development of astrocytes and oligodendrocytes in mice. CD38 is highly expressed in the developing brains between postnatal day 14 (P14) and day 28 (P28). In situ hybridization and FACS analysis revealed that CD38 is expressed predominantly in astrocytes in these periods. Analyses of the cortex of Cd38 knockout (Cd38

Pressure drops and temperature rises caused by the cooling with liquid nitrogen are evaluated for two typical examples of HTS cables. One is the three-in-one-type AC cable located inside a corrugated pipe, and the other is the single-core-type DC cable located inside a straight pipe. The theoretical expressions of pressure drop and temperature rise indicate the contributions from three components such as the friction between the liquid nitrogen and the wetted surfaces, the ratio of the heat input to the mass flow rate of liquid nitrogen and the vertical change of position along the cooling pipe. The numerically estimated results are validated by comparing them with the test results carried out by the other groups. Furthermore, the maximum feeding distance of HTS cable can be determined by adjusting the flow rate of pump if the maximum permissible pressure drop and temperature rise are fixed in advance.

The development of a liquid nitrogen submerged pump for HTS applications is underway. A cryogenic active magnetic bearing is the main component of the pump. The bearing makes the maintenance interval of the pump longer than 3years such interval is essential for the pump in commercial use of HTS applications such as long HTS cable. The bearing will be set in a HTS motor shaft of the pump. In experiments, a shaft without an HTS motor is used and the bearing was immersed in liquid nitrogen. The rotation of the shaft is made by a high-speed motor at room temperature. The bearing consists of two radial bearings and one axial bearing. The shaft was floating by the bearing and was rotated by the motor at room temperature. The steady rotation speed of 2,000, 3,000, 4,000 and 5,000 rpm were achieved, and the rotation speed satisfied the design speed of the pump which is 5,000rpm.

Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disorder caused by the expansion of a polyglutamine tract in the ataxin-1 protein. To date, no fundamental treatments for SCA1 have been elucidated. However, some studies have shown that mesenchymal stem cells (MSCs) are partially effective in other genetic mouse models of cerebellar ataxia. In this study, we tested the efficacy of the intrathecal injection of MSCs in the treatment of SCA1 in transgenic (SCA1-Tg) mice. We found that intrathecal injection of only 3 × 10(3) MSCs greatly mitigated the cerebellar neuronal disorganization observed in SCA1 transgenic mice (SCA1-Tg mice). Although the Purkinje cells (PCs) of 24-week-old nontreated SCA1-Tg mice displayed a multilayer arrangement, SCA1-Tg mice at a similar age injected with MSCs displayed monolayer PCs. Furthermore, intrathecal injection of MSCs suppressed the atrophy of PC dendrites in SCA1-Tg mice. Finally, behavioral tests demonstrated that MSCs normalized deficits in motor coordination in SCA1-Tg mice. Future studies should be performed to develop optimal protocols for intrathecal transplantation of MSCs in SCA1 model primates with the aim of developing applications for SCA1 patients.

During voluntary limb movements, humans exert anticipatory postural adjustments (APAs) to prevent any upcoming equilibrium disturbance that might be provoked by limb movements. Dysfunction in generation or control of APAs is associated with postural deficits in some human patients with cerebellar damage. To examine the role of the cerebellum in APAs, we investigated a conditional transgenic mouse of spinocerebellar ataxia type 3 (SCA3Tg) that has defective cerebellar Purkinje cells. Kinematic analyses and monitoring of electromyographic activities during quadrupedal standing showed that SCA3Tg mice exhibited greater hindlimb instability than wild-type (WT) mice. This instability increased during a reaching task that required postural adjustments associated with voluntary neck movements. Normally, the activities of the hindlimb muscles are synchronized with those in the neck that are the agonists for movement of the head in this reaching task; however, in SCA3Tg mice, activities in the hindlimbs were markedly delayed compared to the neck. These observations cannot simply be explained as a secondary outcome of the muscle atrophy that occurs in SCA3Tg mice. In WT mice with muscle atrophy induced by immobilization of the hindlimbs, we did not find impairment of APAs. These findings suggest that the deficits in APAs during the reaching task in SCA3Tg mice were not due to muscle atrophy in the hindlimbs, but were mainly caused by cerebellar degeneration. Therefore, we conclude that the cerebellum is critically involved in APAs.

SummaryNeural circuits are shaped by activity-dependent elimination of redundant synapses during postnatal development. In many systems, postsynaptic activity is known to be crucial, but the precise mechanisms remain elusive. Here, we report that the immediate early gene Arc/Arg3.1 mediates elimination of surplus climbing fiber (CF) to Purkinje cell (PC) synapses in the developing cerebellum. CF synapse elimination was accelerated when activity of channelrhodopsin-2-expressing PCs was elevated by 2-day photostimulation. This acceleration was suppressed by PC-specific knockdown of either the P/Q-type voltage-dependent Ca2+ channels (VDCCs) or Arc. PC-specific Arc knockdown had no appreciable effect until around postnatal day 11 but significantly impaired CF synapse elimination thereafter, leaving redundant CF terminals on PC somata. The effect of Arc knockdown was occluded by simultaneous knockdown of P/Q-type VDCCs in PCs. We conclude that Arc mediates the final stage of CF synapse elimination downstream of P/Q-type VDCCs by removing CF synapses from PC somata.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by the expansion of polyglutamine repeats in the Ataxin-1 protein. An accumulating body of cerebellar, histological and behavioral analyses has proven that SCA1-knock-in mice (in which the endogenous Atxn1 gene is replaced with mutant Atxn1 that has abnormally expanded 154 CAG repeats) work as a good tool, which resembles the central nervous system pathology of SCA1 patients. However, the peripheral nervous system pathology of the model mice has not been studied despite the fact that the clinical manifestation is also characterized by peripheral involvement. We show here that spinal motor neurons are degenerated in SCA1-knock-in mice. Histologically, some spinal motor neurons of the SCA1-knock-in mice have polyglutamine aggregates in their nuclei and also thinner and demyelinated axons. Electrophysiological examinations of the mice showed slower nerve conduction velocities in spinal motor neurons and lower amplitudes of muscle action potential, compared to wild-type mice. Consistently, the mice displayed decrease in rearing number and total rearing time. These results suggest that the knock-in mice serve as a definite model that reproduces peripheral involvement and are therefore useful for research on the peripheral nervous system pathology in SCA1 patients.

A 16-kDa proteolipid, mediatophore, in Torpedo electric organs mediates Ca 2+-dependent acetylcholine release. Mediatophore is identical to the pore-forming stalk c-subunit of the V0 sector of vacuolar proton ATPase (ATP6V0C). The function of ATP6V0C in the mammalian central nervous system is not clear. Here, we report transfection of adeno-associated viral vectors harboring rat ATP6V0C into the mouse substantia nigra, in which high potassium stimulation increased overflow of endogenous dopamine (DA) measured in the striatum by in vivo microdialysis. Next, in the striatum of 6-hydroxydopamine- lesioned mice, a model of Parkinson's disease (PD), human tyrosine hydroxylase, aromatic l-amino-acid decarboxylase and guanosine triphosphate cyclohydrolase 1, together with or without ATP6V0C, were expressed in the caudoputamen for rescue. Motor performance on the accelerating rotarod test and amphetamine-induced ipsilateral rotation were improved in the rescued mice coexpressing ATP6V0C. [ 3H]DA, taken up into cultured N18 neuronal tumor cells transformed to express ATP6V0C, was released by potassium stimulation. These results indicated that ATP6V0C mediates DA release from nerve terminals in the striatum of DA neurons of normal mice and from gene-transferred striatal cells of parkinsonian mice. The results suggested that ATP6V0C may be useful as a rescue molecule in addition to DA-synthetic enzymes in the gene therapy of PD. © 2011 Elsevier Ltd. All rights reserved.

Fragile X mental retardation protein (FMRP) is absent or highly reduced in Fragile X Syndrome, a genetic disorder causing cognitive impairment and autistic behaviors. Previous proof-of-principle studies have demonstrated that restoring FMRP in the brain using viral vectors can improve pathological abnormalities in mouse models of fragile X. However, unlike small molecule drugs where the dose can readily be adjusted during treatment, viral vector-based biological therapeutic drugs present challenges in terms of achieving optimal dosing and expression levels. The objective of this study was to investigate the consequences of expressing varying levels of FMRP selectively in neurons of Fmr1 knockout and wild-type (WT) mice. A wide range of neuronal FMRP transgene levels was achieved in individual mice after intra-cerebroventricular administration of adeno-associated viral vectors coding for FMRP. In all treated knockout mice, prominent FMRP transgene expression was observed in forebrain structures, whereas lower levels were present in more caudal regions of the brain. Reduced levels of the synaptic protein PSD-95, elevated levels of the transcriptional modulator MeCP2, and abnormal motor activity, anxiety, and acoustic startle responses in Fmr1 knockout mice were fully or partially rescued after expression of FMRP at about 35-115% of WT expression, depending on the brain region examined. In the WT mouse, moderate FMRP over-expression of up to about twofold had little or no effect on PSD-95 and MeCP2 levels or on behavioral endophenotypes. In contrast, excessive over-expression in the Fmr1 knockout mouse forebrain (approximately 2.5-6-fold over WT) induced pathological motor hyperactivity and suppressed the startle response relative to WT mice. These results delineate a range of FMRP expression levels in the central nervous system that confer phenotypic improvement in fragile X mice. Collectively, these findings are pertinent to the development of long-term curative gene therapy strategies for treating Fragile X Syndrome and other neurodevelopmental disorders.

Neuron-specific enolase (NSE) is a glycolytic isoenzyme found in mature neurons and cells of neuronal origin. Injecting adeno-associated virus serotype 9 (AAV9) vectors carrying the NSE promoter into the cerebellar cortex is likely to cause the specific transduction of neuronal cells, such as Purkinje cells (PCs) and interneurons, but not Bergmann glia (BG). However, we found BG-predominant transduction without PC transduction along a traumatic needle tract for viral injection. The enhancement of neuroinflammation by the co-application of lipopolysaccharide (LPS) with AAV9 significantly expanded the BG-predominant area concurrently with the potentiated microglial activation. The BG-predominant transduction was gradually replaced by the PC-predominant transduction as the neuroinflammation dissipated. Experiments using glioma cell cultures revealed significant activation of the NSE promoter due to glucose deprivation, suggesting that intracellularly stored glycogen is metabolized through the glycolytic pathway for energy. Activation of the glycolytic enzyme promoter in BG concurrently with inactivation in PC may have pathophysiological significance for the production of lactate in activated BG and the utilization of lactate, which is provided by the BG-PC lactate shuttle, as a primary energy resource in injured PCs.

Machado-Joseph disease (MJD) is a neurodegenerative spinocerebellar ataxia (SCA) associated with an expanded polyglutamine tract within ataxin-3 for which there is currently no available therapy. We previously showed that caffeine, a nonselective adenosine receptor antagonist, delays the appearance of striatal damage resulting from expression of full-length mutant ataxin-3. Here we investigated the ability of caffeine to alleviate behavioral deficits and cerebellar neuropathology in transgenic mice with a severe ataxia resulting from expression of a truncated fragment of polyglutamine-expanded ataxin-3 in Purkinje cells.Control and transgenic c57Bl6 mice expressing in the mouse cerebella a truncated form of human ataxin-3 with 69 glutamine repeats were allowed to freely drink water or caffeinated water (1g/L). Treatments began at 7 weeks of age, when motor and ataxic phenotype emerges in MJD mice, and lasted up to 20 weeks. Mice were tested in a panel of locomotor behavioral paradigms, namely rotarod, beam balance and walking, pole, and water maze cued-platform version tests, and then sacrificed for cerebellar histology.Caffeine consumption attenuated the progressive loss of general and fine-tuned motor function, balance, and grip strength, in parallel with preservation of cerebellar morphology through decreasing the loss of Purkinje neurons and the thinning of the molecular layer in different folia. Caffeine also rescued the putative striatal-dependent executive and cognitive deficiencies in MJD mice.Our findings provide the first in vivo demonstration that caffeine intake alleviates behavioral disabilities in a severely impaired animal model of SCA. Ann Neurol 2017;81:407-418.

Resistant glucan (RG) is a water-soluble polysaccharide resistant to hydrolysis by digestive enzymes in the human gastrointestinal system. RG mixture (RGM) contains more than 75% RG as dietary fiber and other saccharides. The effects of ingestion of 3.3, 6.6, and 13.2 g/d of RGM (containing of 2.5, 5.0, and 10.0 g/d RG as dietary fiber) on the fecal properties and the frequency of defecation were investigated in 60 female volunteers with constipation. The study was designed as a randomized, single-blinded, and placebo-controlled parallel-group trial. Each subject consumed RGM or a placebo (digestible maltodextrin) for 2 wk. Questionnaire data on the effects on bowel movement were analyzed according to defecation days, defecation frequency, fecal volume, fecal shapes, fecal color, fecal odor, and fecal excretory feeling. The results showed significant increases in defecation frequency (p

The autophagy-lysosome protein degradation is further classified into macroautophagy (MA), microautophagy (mA), and chaperone-mediated autophagy (CMA). While MA is involved in various functions and disease pathogenesis, little is known about CMA and mA because of the absence of easy methods to assess their activities. We have recently established a method to assess CMA activity using glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a CMA substrate, and HaloTag (HT) system. Another group has recently identified a mammalian mA pathway, in which substrates are delivered to late endosomes in an heat shock cognate protein (Hsc)70-dependent manner. Because Hsc70 is also involved in CMA, our method would detect both CMA and mA activities. In this study, we attempted to assess CMA and mA activities separately through the siRNA-mediated knockdown of CMA- and mA-related proteins. Knockdown of LAMP2A, a CMA-related protein, and TSG101, an mA-related protein, significantly but only partially decreased the punctate accumulation of GAPDH-HT in AD293 cells and primary cultured rat cortical neurons. Compounds that activate CMA significantly increased GAPDH-HT puncta in TSG101-knockdown cells, but not in LAMP2A-knockdown cells, suggesting that punctate accumulation of GAPDH-HT under LAMP2A- and TSG101-knockdown represents mA and CMA activities, respectively. We succeeded in establishing the method to separately evaluate CMA and mA activities by fluorescence observation.