Akira Machida

Circulating microRNAs in the cerebrospinal fluid of patients with brain diseases

MicroRNAs (miRNAs) are small, noncoding regulatory RNAs that regulate gene expression at the -posttranscriptional level. Although circulating miRNAs in human body fluids have recently been recognized as disease biomarkers, especially in the field of oncology, little is known about the miRNAs in cerebrospinal fluid (CSF). This chapter describes the feasibility of miRNAs in CSF as biomarkers for the diagnosis of brain diseases and the methods of miRNA isolation from CSF.

Shakuyaku-kanzo-to induces pseudoaldosteronism characterized by hypokalemia, rhabdomyolysis, metabolic alkalosis with respiratory compensation, and increased urinary cortisol levels.

BACKGROUND Licorice, the primary ingredient of the Japanese herbal medicine shakuyaku-kanzo-to, can cause pseudoaldosteronism. Thus, shakuyaku-kanzo-to can cause this condition. CASE DESCRIPTION A 79-year-old woman was brought to the emergency room. She had been experiencing general fatigue, numbness in the hands, and weakness in the lower limbs and could not stand up without assistance. She presented with hypokalemia (potassium level, 1.7 mEq/L), increased urinary excretion of potassium (fractional excretion of K, 21.2%), abnormalities on an electrocardiogram (flat T waves in II, III, AVF, and V1-6), rhabdomyolysis (creatine kinase level, 28,376 U/L), myopathy, metabolic alkalosis with respiratory compensation (O(2) flow rate, 2 L/min; pH, 7.473; pco(2), 61.0 mm Hg; po(2), 78.0 mm Hg; HCO(3), 44.1 mmol/L), hypertension (174/93 mm Hg), hyperglycemia (blood glucose level, 200-300 mg/dL), frequent urination, suppressed plasma renin activity (0.1 ng/mL/hour), decreased aldosterone levels (2.6 ng/dL), and increased urinary cortisol levels (600.6 microg/day; reference range, 26.0-187.0 microg/day). CONCLUSIONS In this case, the observed reduction in the urinary cortisol levels, from 600.6 to 37.8 microg/day, led to a definitive diagnosis of pseudoaldosteronism instead of the apparent mineralocorticoid excess syndrome. Discontinuing shakuyaku-kanzo-to treatment and administering spironolactone and potassium proved effective in improving the patients condition. Medical practitioners prescribing shakuyaku-kanzo-to should take into account the association between licorice, which is its main ingredient, and pseudoaldosteronism.

Intrathecal shRNA-AAV9 Inhibits Target Protein Expression in the Spinal Cord and Dorsal Root Ganglia of Adult Mice

Gene therapy for neurological diseases requires efficient gene delivery to target tissues in the central and peripheral nervous systems. Although adeno-associated virus is one of the most promising vectors for clinical use against neurological diseases, it is difficult to get it across the blood-brain barrier. A clinically practical approach to using a vector based on adeno-associated virus to decrease the expression of a specific gene in both the central and the peripheral nervous system has yet to be established. Here, we analyzed whether upper lumbar intrathecal administration of a therapeutic vector incorporating adeno-associated virus and short-hairpin RNA against superoxide dismutase-1 bypassed the blood-brain barrier to target the spinal cord and dorsal root ganglia. The therapeutic vector effectively suppressed mRNA and protein expression of endogenous superoxide dismutase-1 in the lumbar spinal cord and dorsal root ganglia. Moreover, neither neurological side effects nor toxicity due to the incorporated short-hairpin RNA occurred after the injection. We propose that this approach could be developed into novel therapies for motor neuron diseases and chronic pain conditions, such as complex regional pain syndrome, through silencing of the genes responsible for pathologies in the spinal cord and dorsal root ganglia.

Next-generation sequencing-based small RNA profiling of cerebrospinal fluid exosomes

MicroRNAs (miRNAs), particularly those found in human body fluids, have been suggested as potential biomarkers. Among various body fluids, the cerebrospinal fluid (CSF) shows promise as a profiling target for diagnosis and monitoring of neurological diseases. However, relevant genome-scale studies are limited and no studies have profiled exosomal miRNAs in CSF. Therefore, we conducted a next-generation sequencing-based genome-wide survey of small RNAs in the exosomal and non-exosomal (supernatant) fractions of healthy human CSF as well as serum in each donor. We observed miRNA enrichment in the exosomal fractions relative to the supernatant fractions of both CSF and serum. We also observed substantial differences in exosomal miRNA profiles between CSF and serum. Half of the reported brain miRNAs were found in CSF exosomal fractions. In particular, miR-1911-5p, specifically expressed in brain tissue, was detected in CSF but not in serum, as confirmed by digital PCR in three additional donors. Our data suggest that the brain is a major source of CSF exosomal miRNAs. Here we provide the important evidence that exosomal miRNAs in CSF may reflect brain pathophysiology.

Intraperitoneal administration of AAV9-shRNA inhibits target gene expression in the dorsal root ganglia of neonatal mice

BackgroundThere is considerable interest in inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although short interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge, especially by systemic administration. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) by using short hairpin RNA–expressing single-stranded adeno-associated virus 9 (ssAAV9-shRNA).ResultsIntraperitoneal administration of ssAAV9-shRNA to neonatal mice resulted in highly effective and specific silencing of a target gene in DRG. We observed an approximately 80% reduction in target mRNA in the DRG, and 74.7% suppression of the protein was confirmed by Western blot analysis. There were no major side effects, and the suppression effect lasted for more than three months after the injection of ssAAV9-shRNA.ConclusionsAlthough we previously showed substantial inhibition of target gene expression in DRG via intrathecal ssAAV9-shRNA administration, here we succeeded in inhibiting target gene expression in DRG neurons via intraperitoneal injection of ssAAV9-shRNA. AAV9-mediated delivery of shRNA will pave the way for creating animal models for investigating the molecular biology of the mechanisms of pain and sensory ganglionopathies.

Metastatic CNS lymphoma presenting with periventricular dissemination — MRI and neuropathological findings in an autopsy case

Metastatic CNS lymphoma usually manifests as pachymeningeal or leptomeningeal infiltrates, and periventricular dissemination is rare. A 70-year old man first noticed a mass in the left supraclavicular fossa, and then presented with bilateral parkinsonism, followed by consciousness disturbance. Fluid attenuated inversion recovery (FLAIR) image of brain MRI demonstrated hyperintensities at the parenchyma around the lateral ventricle, third ventricle, and fourth ventricle. Gadolinium-enhanced T1-weighted image demonstrated enhancement along the whole wall of the ventricle. Biopsy of the left supraclavicular lymph nodes established a diagnosis of diffuse large B-cell lymphoma. The patient died of multiple organ failure about 5 months after the onset. Autopsy disclosed periventricular dissemination of lymphoma cells that was most severe around the lateral ventricle. We considered that the lymphoma cells entered the ventricular system through the choroid plexus of the lateral ventricle, followed by dissemination of the periventricular parenchyma.

Tremor in Klinefelter’s syndrome improved by testosterone administration

Tremor in Klinefelter’s syndrome is believed to be essential tremor since the publication of “Klinefelter’s syndrome and essential tremor” in 1969. However, the author also stated that tremor in Klinefelter’s syndrome might differ from essential tremor. A 71-year-old man with Klinefelter’s syndrome who suffers from postural hand tremor is described. The electromyogram indicated lower motor neuron disturbance and chronic neurogenic change. The muscle biopsy indicated neurogenic muscle atrophy. Upon testosterone administration, the amplitude of tremor was reduced and a gradual improvement in handwriting was observed. The tremor in this patient was different from essential tremor. The foresight by Baughman in 1969 proved to be true in this patient. This case report provides new insights into the pathogenesis and treatment of tremor in Klinefelter’s syndrome, which would benefit patients who suffer from the tremor.

Sialidosis type I with neoplasms in siblings: the first clinical cases.

In a family where three of four siblings expressed mutants of NEU1, compound heterozygosity of 649G-to-A and 727G-to-A in the lysosomal neuraminidase (sialidase) gene, responsible for decreased sialidase activity [1], was linked not only to the sialidosis type I phenotype, but also to the occurrence of neoplasms of different origins. This is the first clinical observation that suggests a possible link between the decreased activity of lysosomal sialidase and the development of a variety of neoplasms. The pedigree tree of this family is shown in Fig. 1. Except for patients 1–3, no other family members had sialidosis, and except for patients 1–3, no other family members had any kind of malignancy. There are no known environmental conditions that may facilitate the neoplasm development in their birthplace. In lymphocytes, sialidase activity was significantly decreased in patient 1 (6.8% of control mean), patient 2 (12.5% of control mean) and patient 3 (17.0% of control mean). Their mother had sialidase activity 55.8% of control mean [2]. The activities of b-galactosidase and b-hexosaminidase were within the normal range in these patients. The clinical features of patients, including the neurologic symptoms are shown in Table 1. Four different mammalian sialidases have been identified: Neu1, Neu2, Neu3, and Neu4 [3]. Some mutations in the sialidase gene NEU1 lead to a decrease in the activity of sialidase; however, homozygous or, more frequently, compound heterozygous mutations are necessary for the expression of a phenotype recognized as sialidosis type I, which has autosomal recessive inheritance and is characterized by a progressive lysosomal storage of sialylated glycopeptides and oligosaccharides [4]. The current communication includes the findings obtained from an autopsied patient (patient 2) with sialidosis type I, who died due to intractable lymphoma at the age of 32 years [5]. Because the emergence of neoplasm in this sialidosis patient is the first and the only one in literature, this comorbitidy was initially considered coincidental. Subsequent development of other kinds of neoplasms in his siblings (patients 1 and 3), however, could not be attributed to only coincidence, considering the extreme rarity of sialidosis. The occurrence of neoplasms in these cases raised the possibility that either biallelic mutations in the relevant gene or the resultant decrease in the sialidase activity is linked to the development of these neoplasms. Indeed, it has been reported that a decreased expression of the Neu1 protein is associated with an enhanced metastatic ability of mouse colon adenocarcinoma cells [6]. The expression level of NEU1 mRNA in human colon cancer tissue is lesser than that in the adjacent non-cancerous mucosa [7]. Because the heterozygous mutation of the genes encoding the proteins of this family leads to a decrease in sialidase activity, this underlying condition might have facilitated the development of neoplasm in these siblings; the development of neoplasms may not necessarily be associated with lysosomal catabolism, but rather linked to cell differentiation, cell growth, and apoptosis [8]. It may also be considered that decreased sialidase activity, which is also detected in galactosialidosis, may also be associated with the development of Y. Yagi (&) A. Machida S. Toru T. Kobayashi Department of Neurology, Nakano General Hospital, Chuo 4-59-16 Nakano, Tokyo 164-8607, Japan e-mail: yyagi-tmd@umin.ac.jp

Vagus nerve palsy caused by varicella zoster virus infection without rash

Sirs: Vagus nerve palsy is a relatively common condition in neurological and otolaryngological practice. It may occur on an idiopathic basis and occasionally follows an upper respiratory tract infection [1]. Viral infections have been suggested as a possible cause [2], and some cases have proved to be associated with herpes simplex virus (HSV) infection [3, 4]. Varicella zoster virus (VZV) is known to cause Ramsay Hunt syndrome [5], but it also causes various combinations of cranial nerve palsies [6, 7]. Here we report a case of VZV-associated vagus nerve palsy without any rash. A 48-year-old woman suffered from hoarseness, dysphagia, and left-sided neck pain of abrupt onset and was referred to our hospital on the second day of the illness. Her past medical history was unLETTER TO THE EDITORS

Utility of Autonomic Function Tests to Differentiate Dementia with Lewy Bodies and Parkinson Disease with Dementia from Alzheimer Disease

Objective: We studied autonomic disturbance in patients with dementia with Lewy bodies (DLB), Parkinson disease with dementia (PDD), Alzheimer disease (AD), to determine whether autonomic function tests can be used to distinguish these disorders. Methods: Autonomic function was tested in 56 patients with DLB, 37 patients with PDD, and 59 patients with AD by using the sympathetic skin response, coefficient of variation in R-R interval, the head-up tilt test, serum norepinephrine concentration, and 123I-meta-iodobenzylguanidine cardiac scintigraphy. Symptoms of autonomic dysfunction, such as constipation, urinary symptoms, and orthostatic hypotension, were also noted. Results: The groups did not differ on baseline characteristics other than those associated with Parkinsonism and dementia. All patients with DLB and PDD had some dysautonomia, whereas rates were much lower for patients with AD (19%). Significantly more DLB and PDD patients than AD patients showed abnormalities on autonomic function tests. Conclusions: Autonomic function tests might be quite useful to distinguish DLB and PDD from AD.