Intermitochondrial signaling regulates the uniform distribution of stationary mitochondria in axons.

In the central nervous system (CNS), many neurons develop axonal arbors that are crucial for information processing. Previous studies have demonstrated that premature axons contain motile and stationary mitochondria, and their balance is important for axonal arborization. However, the mechanisms by which neurons determine the positions of stationary mitochondria as well as their turnover remain to be elucidated. We observed that the distribution of stationary mitochondrial spots along the unmyelinated and nonsynaptic axons is not random but rather relatively uniform both in primary cultured neurons and in tissues. Intriguingly, whereas the positions of each mitochondrial spot changed over time, the overall distribution remained uniform. In addition, local inactivation of mitochondria by KillerRed mediated chromophore-assisted light inactivation (CALI) inhibited the translocation of mitochondrial spots in adjacent axonal regions, suggesting that functional mitochondria enhance the motility of other mitochondria in the vicinity. Signals of ATP:ADP sensor, PercevalHR indicated that the ATP:ADP ratio was relatively high around mitochondria, and treating axons with phosphocreatine (PCr), which supplies ATP, reduced the immobile mitochondria induced by the local mitochondrial inactivation. In a mathematical model, we found that the ATP gradient generated by mitochondria, and ATP dependent regulation of mitochondrial motility could establish uniform mitochondrial distribution. These observations suggest that axons in the CNS possess the system that distributes mitochondria uniformly, and intermitochondrial signaling contribute to the regulation. In addition, our results suggest the possibility that ATP might be one of the molecules mediating the signaling.

Optic Nerve Injury Enhanced Mitochondrial Fission and Increased Mitochondrial Density without Altering the Uniform Mitochondrial Distribution in the Unmyelinated Axons of Retinal Ganglion Cells in a Mouse Model.

Glaucomatous optic neuropathy (GON), a major cause of blindness, is characterized by the loss of retinal ganglion cells (RGCs) and the degeneration of their axons. Mitochondria are deeply involved in maintaining the health of RGCs and their axons. Therefore, lots of attempts have been made to develop diagnostic tools and therapies targeting mitochondria. Recently, we reported that mitochondria are uniformly distributed in the unmyelinated axons of RGCs, possibly owing to the ATP gradient. Thus, using transgenic mice expressing yellow fluorescent protein targeting mitochondria exclusively in RGCs within the retina, we assessed the alteration of mitochondrial distributions induced by optic nerve crush (ONC) via in vitro flat-mount retinal sections and in vivo fundus images captured with a confocal scanning ophthalmoscope. We observed that the mitochondrial distribution in the unmyelinated axons of survived RGCs after ONC remained uniform, although their density increased. Furthermore, via in vitro analysis, we discovered that the mitochondrial size is attenuated following ONC. These results suggest that ONC induces mitochondrial fission without disrupting the uniform mitochondrial distribution, possibly preventing axonal degeneration and apoptosis. The in vivo visualization system of axonal mitochondria in RGCs may be applicable in the detection of the progression of GON in animal studies and potentially in humans.

Magnetic Resonance-Guided Focused Ultrasound Thalamotomy for Focal Hand Dystonia: A Pilot Study.

BACKGROUND: The efficacy of magnetic resonance-guided focused ultrasound (MRgFUS) thalamotomy for the treatment of focal hand dystonia (FHD) is not well known. OBJECTIVE: We aimed to prospectively investigate the efficacy of MRgFUS thalamotomy for the treatment of FHD. METHODS: We performed MRgFUS thalamotomy of the ventro-oral (Vo) nucleus in 10 patients with FHD. We evaluated the scores of the Writer's Cramp Rating Scale (WCRS, 0-30; higher scores indicating greater severity), Tubiana Musician's Dystonia Scale (TMDS, 0-5; lower scores indicating greater severity), and Arm Dystonia Disability Scale (ADDS, 0%-100%; lower scores indicating greater disability) at baseline and 3 and 12 months post-treatment. RESULTS: WCRS, TMDS, and ADDS scores significantly improved from 6.3 ± 2.7, 1.4 ± 0.5, and 58.7% ± 14.3% at baseline to 1.6 ± 3.1 (P = 0.011), 5.0 ± 0 (P = 0.0001), and 81.6% ± 22.9% (P = 0.0229) at 12 months, respectively. There was one prolonged case of dysarthria at 12 months. CONCLUSION: We show that MRgFUS Vo-thalamotomy significantly improved FHD. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Kinesin-1 sorting in axons controls the differential retraction of arbor terminals.

The ability of neurons to generate multiple arbor terminals from a single axon is crucial for establishing proper neuronal wiring. Although growth and retraction of arbor terminals are differentially regulated within the axon, the mechanisms by which neurons locally control their structure remain largely unknown. In the present study, we found that the kinesin-1 (Kif5 proteins) head domain (K5H) preferentially marks a subset of arbor terminals. Time-lapse imaging clarified that these arbor terminals were more stable than others, because of a low retraction rate. Local inhibition of kinesin-1 in the arbor terminal by chromophore-assisted light inactivation (CALI) enhanced the retraction rate. The microtubule turnover was locally regulated depending on the length from the branching point to the terminal end, but did not directly correlate with the presence of K5H. By contrast, F-actin signal values in arbor terminals correlated spatiotemporally with K5H, and inhibition of actin turnover prevented retraction. Results from the present study reveal a new system mediated by kinesin-1 sorting in axons that differentially controls stability of arbor terminals.

Ttll9-/- mice sperm flagella show shortening of doublet 7, reduction of doublet 5 polyglutamylation and a stall in beating.

Nine outer doublet microtubules in axonemes of flagella and cilia are heterogeneous in structure and biochemical properties. In mammalian sperm flagella, one of the factors to generate the heterogeneity is tubulin polyglutamylation, although the importance of the heterogeneous modification is unclear. Here, we show that a tubulin polyglutamylase Ttll9 deficiency (Ttll9(-/-)) causes a unique set of phenotypes related to doublet heterogeneity. Ttll9(-/-) sperm axonemes had frequent loss of a doublet and reduced polyglutamylation. Intriguingly, the doublet loss selectively occurred at the distal region of doublet 7, and reduced polyglutamylation was observed preferentially on doublet 5. Ttll9(-/-) spermatozoa showed aberrant flagellar beating, characterized by frequent stalls after anti-hook bending. This abnormal motility could be attributed to the reduction of polyglutamylation on doublet 5, which probably occurred at a position involved in the switching of bending. These results indicate that mammalian Ttll9 plays essential roles in maintaining the normal structure and beating pattern of sperm flagella by establishing normal heterogeneous polyglutamylation patterns.

DBZ regulates cortical cell positioning and neurite development by sustaining the anterograde transport of Lis1 and DISC1 through control of Ndel1 dual-phosphorylation.

Cell positioning and neuronal network formation are crucial for proper brain function. Disrupted-in-Schizophrenia 1 (DISC1) is anterogradely transported to the neurite tips, together with Lis1, and functions in neurite extension via suppression of GSK3ß activity. Then, transported Lis1 is retrogradely transported and functions in cell migration. Here, we show that DISC1-binding zinc finger protein (DBZ), together with DISC1, regulates mouse cortical cell positioning and neurite development in vivo. DBZ hindered Ndel1 phosphorylation at threonine 219 and serine 251. DBZ depletion or expression of a double-phosphorylated mimetic form of Ndel1 impaired the transport of Lis1 and DISC1 to the neurite tips and hampered microtubule elongation. Moreover, application of DISC1 or a GSK3ß inhibitor rescued the impairments caused by DBZ insufficiency or double-phosphorylated Ndel1 expression. We concluded that DBZ controls cell positioning and neurite development by interfering with Ndel1 from disproportionate phosphorylation, which is critical for appropriate anterograde transport of the DISC1-complex.

Study of local intracellular signals regulating axonal morphogenesis using a microfluidic device.

The establishment and maintenance of axonal patterning is crucial for neuronal function. To identify the molecular systems that operate locally to control axonal structure, it is important to manipulate molecular functions in restricted subcellular areas for a long period of time. Microfluidic devices can be powerful tools for such purposes. In this study, we demonstrate the application of a microfluidic device to clarify the function of local Ca2+ signals in axons. Membrane depolarization significantly induced axonal branch-extension in cultured cerebellar granule neurons (CGNs). Local application of nifedipine using a polydimethylsiloxane (PDMS)-based microfluidic device demonstrated that Ca2+ entry from the axonal region via L-type voltage-dependent calcium channels (L-VDCC) is required for branch extension. Furthermore, we developed a method for locally controlling protein levels by combining genetic techniques and use of a microfluidic culture system. A vector for enhanced green fluorescent protein (EGFP) fused to a destabilizing domain derived from E. coli dihydrofolate reductase (ecDHFR) is introduced in neurons by electroporation. By local application of the DHFR ligand, trimethoprim (TMP) using a microfluidic device, we were able to manipulate differentially the level of fusion protein between axons and somatodendrites. The present study revealed the effectiveness of microfluidic devices to address fundamental biological issues at subcellular levels, and the possibility of their development in combination with molecular techniques.

Axonal gradient of arachidonic acid-containing phosphatidylcholine and its dependence on actin dynamics.

Phosphatidylcholine (PC) is the most abundant component of lipid bilayers and exists in various molecular forms, through combinations of two acylated fatty acids. Arachidonic acid (AA)-containing PC (AA-PC) can be a source of AA, which is a crucial mediator of synaptic transmission and intracellular signaling. However, the distribution of AA-PC within neurons has not been indicated. In the present study, we used imaging mass spectrometry to characterize the distribution of PC species in cultured neurons of superior cervical ganglia. Intriguingly, PC species exhibited a unique distribution that was dependent on the acyl chains at the sn-2 position. In particular, we found that AA-PC is enriched within the axon and is distributed across a proximal-to-distal gradient. Inhibitors of actin dynamics (cytochalasin D and phallacidin) disrupted this gradient. This is the first report of the gradual distribution of AA-PC along the axon and its association with actin dynamics.

Usefulness of Leksell GammaPlan for preoperative planning of brain tumor resection: delineation of the cranial nerves and fusion of the neuroimaging data, including diffusion tensor imaging.

Leksell GammaPlan (LGP) software was initially designed for Gamma Knife radiosurgery, but it can be successfully applied to planning of the open neurosurgical procedures as well. We present our initial experience of delineating the cranial nerves in the vicinity of skull base tumors, combined visualization of the implanted subdural electrodes and cortical anatomy to facilitate brain mapping, and fusion of structural magnetic resonance imaging and diffusion tensor imaging performed with the use of LGP before removal of intracranial neoplasms. Such preoperative information facilitated choosing the optimal approach and general surgical strategy, and corresponded well to the intraoperative findings. Therefore, LGP may be helpful for planning open neurosurgical procedures in cases of both extraaxial and intraaxial intracranial tumors.

A model for generating differences in microtubules between axonal branches depending on the distance from terminals.

In the remodeling of axonal arbor, the growth and retraction of branches are differentially regulated within a single axon. Although cell-autonomously generated differences in microtubule (MT) turnover are thought to be involved in selective branch regulation, the cellular system whereby neurons generate differences of MTs between axonal branches has not been clarified. Because MT turnover tends to be slower in longer branches compared with neighboring shorter branches, feedback regulation depending on branch length is thought to be involved. In the present study, we generated a model of MT lifetime in axonal terminal branches by adapting a length-dependent model in which parameters for MT dynamics were constant in the arbor. The model predicted that differences in MT lifetime between neighboring branches could be generated depending on the distance from terminals. In addition, the following points were predicted. Firstly, destabilization of MTs throughout the arbor decreased the differences in MT lifetime between branches. Secondly, differences of MT lifetime existed even before MTs entered the branch point. In axonal MTs in primary neurons, treatment with a low concentration of nocodazole significantly decreased the differences of detyrosination (deTyr) and tyrosination (Tyr) of tubulins, indicators of MT turnover. Expansion microscopy of the axonal shaft before the branch point revealed differences in deTyr/Tyr modification on MTs. Our model recapitulates the differences in MT turnover between branches and provides a feedback mechanism for MT regulation that depends on the axonal arbor geometry.

Tubulin Polyglutamylation by TTLL1 and TTLL7 Regulate Glutamate Concentration in the Mice Brain.

As an important neurotransmitter, glutamate acts in over 90% of excitatory synapses in the human brain. Its metabolic pathway is complicated, and the glutamate pool in neurons has not been fully elucidated. Tubulin polyglutamylation in the brain is mainly mediated by two tubulin tyrosine ligase-like (TTLL) proteins, TTLL1 and TTLL7, which have been indicated to be important for neuronal polarity. In this study, we constructed pure lines of Ttll1 and Ttll7 knockout mice. Ttll knockout mice showed several abnormal behaviors. Matrix-assisted laser desorption/ionization (MALDI) Imaging mass spectrometry (IMS) analyses of these brains showed increases in glutamate, suggesting that tubulin polyglutamylation by these TTLLs acts as a pool of glutamate in neurons and modulates some other amino acids related to glutamate.

Identification of tubulin deglutamylase among Caenorhabditis elegans and mammalian cytosolic carboxypeptidases (CCPs).

Tubulin polyglutamylation is a reversible post-translational modification, serving important roles in microtubule (MT)-related processes. Polyglutamylases of the tubulin tyrosine ligase-like (TTLL) family add glutamate moieties to specific tubulin glutamate residues, whereas as yet unknown deglutamylases shorten polyglutamate chains. First we investigated regulatory machinery of tubulin glutamylation in MT-based sensory cilia of the roundworm Caenorhabditis elegans. We found that ciliary MTs were polyglutamylated by a process requiring ttll-4. Conversely, loss of ccpp-6 gene function, which encodes one of two cytosolic carboxypeptidases (CCPs), resulted in elevated levels of ciliary MT polyglutamylation. Consistent with a deglutamylase function for ccpp-6, overexpression of this gene in ciliated cells decreased polyglutamylation signals. Similarly, we confirmed that overexpression of murine CCP5, one of two sequence orthologs of nematode ccpp-6, caused a dramatic loss of MT polyglutamylation in cultured mammalian cells. Finally, using an in vitro assay for tubulin glutamylation, we found that recombinantly expressed Myc-tagged CCP5 exhibited deglutamylase biochemical activities. Together, these data from two evolutionarily divergent systems identify C. elegans CCPP-6 and its mammalian ortholog CCP5 as a tubulin deglutamylase.

Cell-intrinsic regulation of axonal morphogenesis by the Cdh1-APC target SnoN.

Axonal growth is fundamental to the establishment of neuronal connectivity in the brain. However, the cell-intrinsic mechanisms that govern axonal morphogenesis remain to be elucidated. The ubiquitin ligase Cdh1-anaphase-promoting complex (Cdh1-APC) suppresses the growth of axons in postmitotic neurons. Here, we report that Cdh1-APC operates in the nucleus to inhibit axonal growth. We also identify the transcriptional corepressor SnoN as a key target of neuronal Cdh1-APC that promotes axonal growth. Cdh1 forms a physical complex with SnoN and stimulates the ubiquitin-dependent proteasomal degradation of SnoN in neurons. Knockdown of SnoN in neurons significantly reduces axonal growth and suppresses Cdh1 RNAi enhancement of axonal growth. In addition, SnoN knockdown in vivo suggests an essential function for SnoN in the development of granule neuron parallel fibers in the cerebellar cortex. These findings define Cdh1-APC and SnoN as components of a cell-intrinsic pathway that orchestrates axonal morphogenesis in a transcription-dependent manner in the mammalian brain.

Developments and applications of mass microscopy.

We have developed a mass microscopy technique, i.e., a microscope combined with high-resolution matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), which is a powerful tool for investigating the spatial distribution of biomolecules without any time-consuming extraction, purification, and separation procedures for biological tissue sections. Mass microscopy provides clear images about the distribution of hundreds of biomolecules in a single measurement and also helps in understanding the cellular profile of the biological system. The sample preparation and the spatial resolution and speed of the technique are all important steps that affect the identification of biomolecules in mass microscopy. In this Award Lecture Review, we focus on some of the recent developments in clinical applications to show how mass microscopy can be employed to assess medical molecular morphology.

Reliability of Residual Tumor Estimation Based on Navigation Log.

The mass of residual tumors has previously been estimated using time-series records of the position of surgical instruments acquired from neurosurgical navigation systems (navigation log). This method has been shown to be useful for rapid evaluation of residual tumors during resection. However, quantitative analysis of the method's reliability has not been sufficiently reported. The effect of poor log coverage is dominant in previous studies, in that it did not highlight other disturbance factors, such as intraoperative brain shift. We analyzed 25 patients with a high log-acquisition rate that was calculated by dividing the log-available time by the instrument-use time. We estimated the region of resection using the trajectory of surgical instrument that was extracted from the navigation log. We then calculated the residual tumor region and measured its volume as log-estimation residual tumor volume (RTV). We evaluated the correlation between the log-estimation RTV and the RTV in the post-resection magnetic resonance (MR) image. We also evaluated the accuracy of detecting the residual tumor mass using the estimated residual tumor region. The log-estimation RTV and the RTV in the post-resection MR image were significantly correlated (correlation coefficient = 0.960; P <0.001). The presence of patient-wise residual tumor mass was detected with a sensitivity of 81.8% and a specificity of 92.9%. The individual residual tumor mass was detected with a positive predictive value of 72%. Estimation of residual tumor with adequate log coverage appears to be a suitable method with a high reliability. This method can support rapid decision-making during resection.

Neuroprotective effects of aromatic turmerone on activity deprivation-induced apoptosis in cerebellar granule neurons.

Ar-turmerone, which is a major bioactive component found in the essential oil derived from Curcuma longa, has been reported to inhibit proliferation and induce apoptosis in cancer cell lines. Recently, ar-turmerone has been reported to increase the proliferation of neuronal stem cells, in contrast to its actions in cancer cells. These observations raise the possibility that ar-turmerone serves specific functions in neuronal cell lineages. However, the effects of ar-turmerone on postmitotic neurons remain elusive. In the present study, we investigated the neuroprotective functions of ar-turmerone in primary cerebellar granule neuronal cultures. We found that ar-turmerone increased the survival of neurons following activity deprivation. Consistently, the induction of cleaved caspase-3, a hallmark of apoptosis, was prevented by ar-turmerone, although neither the level of reactive oxygen species nor the mitochondrial membrane potential was affected. This study reports a neuroprotective function for ar-turmerone, providing new insights into the potential therapeutic applications of ar-turmerone for neurological disorders.

TGFbeta-Smad2 signaling regulates the Cdh1-APC/SnoN pathway of axonal morphogenesis.

Axon growth is critical to the establishment of neuronal connectivity. The E3 ubiquitin ligase Cdh1-anaphase-promoting complex (Cdh1-APC) and its substrate the transcriptional modulator SnoN form a cell-intrinsic pathway that orchestrates axonal morphogenesis in the mammalian brain. How the Cdh1-APC/SnoN pathway is controlled in the nervous system remained unknown. Here, we report that the TGFbeta-regulated signaling protein Smad2 plays a key role in regulating the Cdh1-APC/SnoN pathway in neurons. We find that Smad2 is expressed in primary granule neurons of the developing rat cerebellar cortex. The Smad signaling pathway is basally activated in neurons. Endogenous Smad2 is phosphorylated, localized in the nucleus, and forms a physical complex with endogenous SnoN in granule neurons. Inhibition of Smad signaling by several distinct approaches, including genetic knock-down of Smad2, stimulates axonal growth. Biochemical evidence and genetic epistasis analyses reveal that Smad2 acts upstream of SnoN in a shared pathway with Cdh1-APC in the control of axonal growth. Remarkably, Smad2 knock-down also overrides the ability of adult rat myelin to inhibit axonal growth. Collectively, our findings define a novel function for Smad2 in regulation of the Cdh1-APC/SnoN cell-intrinsic pathway of axonal morphogenesis, and suggest that inhibition of Smad signaling may hold therapeutic potential in stimulating axonal growth after injury in the CNS.

Visualization of the cell-selective distribution of PUFA-containing phosphatidylcholines in mouse brain by imaging mass spectrometry.

Previous studies have shown that MALDI-imaging mass spectrometry (IMS) can be used to visualize the distribution of various biomolecules, especially lipids, in the cells and tissues. In this study, we report the cell-selective distribution of PUFA-containing glycerophospholipids (GPLs) in the mouse brain. We established a practical experimental procedure for the IMS of GPLs. We demonstrated that optimization of the composition of the matrix solution and spectrum normalization to the total ion current (TIC) is critical. Using our procedure, we simultaneously differentiated and visualized the localizations of specific molecular species of GPLs in mouse brain sections. The results showed that PUFA-containing phosphatidylcholines (PCs) were distributed in a cell-selective manner: arachidonic acid- and docosahexaenoic acid-containing PCs were seen in the hippocampal neurons and cerebellar Purkinje cells, respectively. Furthermore, these characteristic localizations of PUFA-PCs were formed during neuronal maturation. The phenomenon of brain cell-selective production of specific PUFA-GPLs will help elucidate the potential physiological functions of PUFAs in specific brain regions.

Correlation between fractional anisotropy changes in the targeted ventral intermediate nucleus and clinical outcome after transcranial MR-guided focused ultrasound thalamotomy for essential tremor: results of a pilot study.

OBJECTIVE: This study evaluated changes of fractional anisotropy (FA) in the ventral intermediate nucleus (VIM) of the thalamus after transcranial MR-guided focused ultrasound (TcMRgFUS) thalamotomy and their associations with clinical outcome. METHODS: Clinical and radiological data of 12 patients with medically refractory essential tremor (mean age 76.5 years) who underwent TcMRgFUS thalamotomy with VIM targeting were analyzed retrospectively. The Clinical Rating Scale for Tremor (CRST) score was calculated before and at 1 year after treatment. Measurements of the relative FA (rFA) values, defined as ratio of the FA value in the targeted VIM to the FA value in the contralateral VIM, were performed before thalamotomy, and 1 day and 1 year thereafter. RESULTS: TcMRgFUS thalamotomy was well tolerated and no long-term complications were noted. At 1-year follow-up, 8 patients demonstrated relief of tremor (improvement group), whereas in 4 others persistent tremor was noted (recurrence group). In the entire cohort, mean rFA values in the targeted VIM before treatment, and at 1 day and 1 year after treatment, were 1.12 ± 0.15, 0.44 ± 0.13, and 0.82 ± 0.22, respectively (p < 0.001). rFA values were consistently higher in the recurrence group compared with the improvement group, and the difference reached statistical significance at 1 day (p < 0.05) and 1 year (p < 0.01) after treatment. There was a statistically significant (p < 0.01) positive correlation between rFA values in the targeted VIM at 1 day after thalamotomy and CRST score at 1 year after treatment. Receiver operating characteristic curve analysis revealed that the optimal cutoff value of rFA at 1 day after thalamotomy for prediction of symptomatic improvement at 1-year follow-up is 0.54. CONCLUSIONS: TcMRgFUS thalamotomy results in significant decrease of rFA in the targeted VIM, at both 1 day and 1 year after treatment. Relative FA values at 1 day after treatment showed significant correlation with CRST score at 1-year follow-up. Therefore, FA may be considered a possible imaging biomarker for early prediction of clinical outcome after TcMRgFUS thalamotomy for essential tremor.

Sonodynamic Therapy With Anticancer Micelles and High-Intensity Focused Ultrasound in Treatment of Canine Cancer.

Sonodynamic therapy (SDT) is a minimally invasive anticancer therapy involving a chemical sonosensitizer and high-intensity focused ultrasound (HIFU). SDT enables the reduction of drug dose and HIFU irradiation power compared to those of conventional monotherapies. In our previous study, mouse models of colon and pancreatic cancer were used to confirm the effectiveness of SDT vs. drug-only or HIFU-only therapy. To validate its usefulness, we performed a clinical trial of SDT using an anticancer micelle (NC-6300) and our HIFU system in four pet dogs with spontaneous tumors, including chondrosarcoma, osteosarcoma, hepatocellular cancer, and prostate cancer. The fact that no adverse events were observed, suggests the usefulness of SDT.