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.

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 Ca(2+) 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 Ca(2+) surges are absent. Together, we propose that tDCS changes the metaplasticity of the cortex through astrocytic Ca(2+)/IP3 signalling.

Cerebellar Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex, and damage to PCs results in motor deficits. Spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease), a hereditary neurodegenerative disease, is caused by an abnormal expansion of the polyglutamine tract in the causative ATXN3 protein. SCA3 affects a wide range of cells in the central nervous system, including those in the cerebellum. To unravel SCA3 pathology, we used adeno-associated virus serotype 9 (AAV9) vectors to express full-length ATXN3 with an abnormally expanded 89 polyglutamine stretch (ATXN3[Q89]) in cerebellar neurons of mature wild-type mice. Mice expressing ATXN3[Q89] exhibited motor impairment in a manner dependent on the viral titer. Immunohistochemistry of the cerebellum showed ubiquitinated nuclear aggregates in PCs; degeneration of PC dendrites; and a significant decrease in multiple proteins including retinoid-related orphan receptor α (RORα), a transcription factor, and type 1 metabotropic glutamate receptor (mGluR1) signaling molecules. Patch clamp analysis of ATXN3[Q89]-expressing PCs revealed marked defects in mGluR1 signaling. Notably, the emergence of behavioral, morphological, and functional defects was inhibited by a single injection of SR1078, an RORα/γ agonist. These results suggest that RORα plays a key role in mutant ATXN3-mediated aberrant phenotypes and that the pharmacological enhancement of RORα could function as a method for therapeutic intervention in SCA3.

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.

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.

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. [(3)H]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.

Neural 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 Ca(2+) 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.

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. This expansion translates into a polyglutamine tract that confers a toxic gain-of-function to the mutant protein--ataxin-3, leading to neurodegeneration in specific brain regions, with particular severity in the cerebellum. No treatment able to modify the disease progression is available. However, gene silencing by RNA interference has shown promising results. Therefore, in this study we investigated whether lentiviral-mediated allele-specific silencing of the mutant ataxin-3 gene, after disease onset, would rescue the motor behavior deficits and neuropathological features in a severely impaired transgenic mouse model of MJD. For this purpose, we injected lentiviral vectors encoding allele-specific silencing-sequences (shAtx3) into the cerebellum of diseased transgenic mice expressing the targeted C-variant of mutant ataxin-3 present in 70% of MJD patients. This variation permits to discriminate between the wild-type and mutant forms, maintaining the normal function of the wild-type allele and silencing only the mutant form. Quantitative analysis of rotarod performance, footprint and activity patterns revealed significant and robust alleviation of gait, balance (average 3-fold increase of rotarod test time), locomotor and exploratory activity impairments in shAtx3-injected mice, as compared to control ones injected with shGFP. An important improvement of neuropathology was also observed, regarding the number of intranuclear inclusions, calbindin and DARPP-32 immunoreactivity, fluorojade B and Golgi staining and molecular and granular layers thickness. These data demonstrate for the first time the efficacy of gene silencing in blocking the MJD-associated motor-behavior and neuropathological abnormalities after the onset of the disease, supporting the use of this strategy for therapy of MJD.

The neurobiological basis of autism spectrum disorder (ASD) remains poorly understood. Given the role of CD38 in social recognition through oxytocin (OT) release, we hypothesized that CD38 may play a role in the etiology of ASD. Here, we first examined the immunohistochemical expression of CD38 in the hypothalamus of post-mortem brains of non-ASD subjects and found that CD38 was colocalized with OT in secretory neurons. In studies of the association between CD38 and autism, we analyzed 10 single nucleotide polymorphisms (SNPs) and mutations of CD38 by re-sequencing DNAs mainly from a case-control study in Japan, and Caucasian cases mainly recruited to the Autism Genetic Resource Exchange (AGRE). The SNPs of CD38, rs6449197 (p<0.040) and rs3796863 (p<0.005) showed significant associations with a subset of ASD (IQ>70; designated as high-functioning autism (HFA)) in the U.S. 104 AGRE family trios, but not with Japanese 188 HFA subjects. A mutation that caused tryptophan to replace arginine at amino acid residue 140 (R140W; (rs1800561, 4693C>T)) was found in 0.6-4.6% of the Japanese population and was associated with ASD in the smaller case-control study. The SNP was clustered in pedigrees in which the fathers and brothers of T-allele-carrier probands had ASD or ASD traits. In this cohort OT plasma levels were lower in subjects with the T allele than in those without. One proband with the T allele who was taking nasal OT spray showed relief of symptoms. The two variant CD38 poloymorphysms tested may be of interest with regard of the pathophysiology of ASD.

Glucocorticoids are released from the adrenal cortex and are important for regulating various physiological functions. However, a persistent increase in glucocorticoids due to chronic stress causes various dysfunctions in the central nervous system which can lead to mental disorders such as depression. Macroautophagy, one of the pathways of the autophagy-lysosome protein degradation system, is dysregulated in psychiatric disorders, implicating a disturbance of protein degradation in the pathogenesis of psychiatric disorders. In the present study, we investigated whether glucocorticoids affect the activity of chaperone-mediated autophagy (CMA) and microautophagy (mA), the other two pathways of the autophagy-lysosome system. Treatment of human-derived AD293 cells and primary cultured rat cortical neurons with dexamethasone, a potent glucocorticoid receptor agonist, and endogenous glucocorticoids decreased both CMA and mA activities. However, this decrease was significantly suppressed by treatment with RU-486, a glucocorticoid receptor antagonist. In addition, dexamethasone significantly decreased lysosomal Hsc70. These findings suggest that glucocorticoids negatively regulate CMA and mA in a glucocorticoid receptor-dependent manner, and provide evidence for CMA and mA as novel therapeutic targets for depression.

The adult brain can flexibly adapt behaviors to specific life-stage demands. For example, while sexually naive male mice are aggressive to the conspecific young, they start to provide caregiving to infants around the time when their own young are expected. How such behavioral plasticity is implemented at the level of neural connections remains poorly understood. Here, using viral-genetic approaches, we establish hypothalamic oxytocin neurons as the key regulators of the parental caregiving behaviors of male mice. We then use rabies-virus-mediated unbiased screening to identify excitatory neural connections originating from the lateral hypothalamus to the oxytocin neurons to be drastically strengthened when male mice become fathers. These connections are functionally relevant, as their activation suppresses pup-directed aggression in virgin males. These results demonstrate the life-stage associated, long-distance, and cell-type-specific plasticity of neural connections in the hypothalamus, the brain region that is classically assumed to be hard-wired.

This protocol describes BATTLE-1EX, which is a combined method of BATTLE-1 and expansion microscopy to obtain high-resolution imaging of whole synaptic structures and their components of hippocampal neural circuits. BATTLE-1 uses two genetically engineered recombinase proteins and competition between two recombinases that can be independently titrated, resulting in a tunable proportion of mCherry+/YFP- and YFP+/mCherry- cells. As a combinational method, BATTLE-1EX has the potential to visualize and dissect whole synaptic structures in numerous regions in the brain. For complete details on the use and execution of this protocol, please refer to Kohara et al. (2020).

Chaperone-mediated autophagy (CMA) is a pathway in the autophagy-lysosome protein degradation system. CMA impairment has been implicated to play a role in spinocerebellar ataxia (SCA) pathogenesis. D-cysteine is metabolized by D-amino acid oxidase (DAO), leading to hydrogen sulfide generation in the cerebellum. Although D-cysteine alleviates the disease phenotypes in SCA-model mice, it remains unknown how hydrogen sulfide derived from D-cysteine exerts this effect. In the present study, we investigated the effects of D-cysteine and hydrogen sulfide on CMA activity using a CMA activity marker that we have established. D-cysteine activated CMA in Purkinje cells (PCs) of primary cerebellar cultures where DAO was expressed, while it failed to activate CMA in DAO-deficient AD293 cells. In contrast, Na2S, a hydrogen sulfide donor, activated CMA in both PCs and AD293 cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) is known to be activated by hydrogen sulfide and regulate CMA activity. An Nrf2 inhibitor, ML385, prevented CMA activation triggered by D-cysteine and Na2S. Additionally, long-term treatment with D-cysteine increased the amounts of Nrf2 and LAMP2A, a CMA-related protein, in the mouse cerebellum. These findings suggest that hydrogen sulfide derived from D-cysteine enhances CMA activity via Nrf2 activation.

Upon systemic administration, adeno-associated virus serotype 9 (AAV9) and the capsid variant PHP.eB show distinct tropism for the central nervous system (CNS), whereas AAV2 and the capsid variant BR1 transduce brain microvascular endothelial cells (BMVECs) with little transcytosis. Here, we show that a single amino acid substitution (from Q to N) in the BR1 capsid at position 587 (designated BR1N) confers a significantly higher blood-brain barrier (BBB) penetration capacity to BR1. Intravenously infused BR1N showed significantly higher CNS tropism than BR1 and AAV9. BR1 and BR1N likely use the same receptor for entry into BMVECs; however, the single amino acid substitution has profound consequences on tropism. This suggests that receptor binding alone does not determine the final outcome in vivo and that further improvements of capsids within predetermined receptor usage are feasible.

Appropriately responding to various sensory signals in the environment is essential for animal survival. Accordingly, animal behaviors are closely related to external and internal states, which include the positive and negative emotional values of sensory signals triggered by environmental factors. While the lateral parabrachial nucleus (LPB) plays a key role in nociception and supports negative valences, it also transmits signals including positive valences. However, the downstream neuronal mechanisms of positive and negative valences have not been fully explored. In the present study, we investigated the ventral tegmental area (VTA) as a projection target for LPB neurons. Optogenetic activation of LPB-VTA terminals in male mice elicits positive reinforcement in an operant task and induces both avoidance and attraction in a place-conditioning task. Inhibition of glutamic acid decarboxylase (GAD) 65-expressing cells in the VTA promotes avoidance behavior induced by photoactivation of the LPB-VTA pathway. These findings indicate that the LPB-VTA pathway is one of the LPB outputs for the transmission of positive and negative valence signals, at least in part, with GABAergic modification in VTA.

Mammalian sires participate in infant care. We previously demonstrated that sires of a strain of nonmonogamous laboratory mice initiate parental retrieval behavior in response to olfactory and auditory signals from the dam during isolation in a new environment. This behavior is rapidly lost in the absence of such signals when the sires are caged alone. The neural circuitry and hormones that control paternal behavior are not well-understood. CD38, a membrane glycoprotein, catalyzes synthesis of cyclic ADP-ribose and facilitates oxytocin (OT) secretion due to cyclic ADP-ribose-dependent increases in cytosolic free calcium concentrations in oxytocinergic neurons in the hypothalamus. In this paper, we studied CD38 in the nucleus accumbens (NAcc) and the role of OT on paternal pup retrieval behavior using CD38 knockout (CD38-/-) mice of the ICR strain.

Cell-type-specific promoters in combination with viral vectors and gene-editing technology permit efficient gene manipulation in specific cell populations. Cerebellar Purkinje cells play a pivotal role in cerebellar functions. Although the Purkinje cell-specific L7 promoter is widely used for the generation of transgenic mice, it remains unsuitable for viral vectors because of its large size (3 kb) and exceedingly weak promoter activity. Here, we found that the 0.8-kb region (named here as L7-6) upstream of the transcription initiation codon in the first exon was alone sufficient as a Purkinje cell-specific promoter, presenting a far stronger promoter activity over the original 3-kb L7 promoter with a sustained significant specificity to Purkinje cells. Intravenous injection of adeno-associated virus vectors that are highly permeable to the blood-brain barrier confirmed the Purkinje cell specificity of the L7-6 in the CNS. The features of the L7-6 were also preserved in the marmoset, a non-human primate. The high sequence homology of the L7-6 among mouse, marmoset, and human suggests the preservation of the promoter strength and Purkinje cell specificity features also in humans. These findings suggest that L7-6 will facilitate the cerebellar research targeting the pathophysiology and gene therapy of cerebellar disorders.

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.

Polyglutamine diseases are inherited neurodegenerative diseases caused by the expanded polyglutamine proteins (polyQs). We have identified a novel guanosine triphosphatase (GTPase) named CRAG that contains a nuclear localization signal (NLS) sequence and forms nuclear inclusions in response to stress. After ultraviolet irradiation, CRAG interacted with and induced an enlarged ring-like structure of promyelocytic leukemia protein (PML) body in a GTPase-dependent manner. Reactive oxygen species (ROS) generated by polyQ accumulation triggered the association of CRAG with polyQ and the nuclear translocation of the CRAG-polyQ complex. Furthermore, CRAG promoted the degradation of polyQ at PML/CRAG bodies through the ubiquitin-proteasome pathway. CRAG knockdown by small interfering RNA in neuronal cells consistently blocked the nuclear translocation of polyQ and enhanced polyQ-mediated cell death. We propose that CRAG is a modulator of PML function and dynamics in ROS signaling and is protectively involved in the pathogenesis of polyglutamine diseases.

Abnormal mitochondrial fragmentation by dynamin-related protein1 (Drp1) is associated with the progression of aging-associated heart diseases, including heart failure and myocardial infarction (MI). Here, we report a protective role of outer mitochondrial membrane (OMM)-localized E3 ubiquitin ligase MITOL/MARCH5 against cardiac senescence and MI, partly through Drp1 clearance by OMM-associated degradation (OMMAD). Persistent Drp1 accumulation in cardiomyocyte-specific MITOL conditional-knockout mice induced mitochondrial fragmentation and dysfunction, including reduced ATP production and increased ROS generation, ultimately leading to myocardial senescence and chronic heart failure. Furthermore, ischemic stress-induced acute downregulation of MITOL, which permitted mitochondrial accumulation of Drp1, resulted in mitochondrial fragmentation. Adeno-associated virus-mediated delivery of the MITOL gene to cardiomyocytes ameliorated cardiac dysfunction induced by MI. Our findings suggest that OMMAD activation by MITOL can be a therapeutic target for aging-associated heart diseases, including heart failure and MI.