Nobuhiro Ogawa

Gene therapy for neuropathic pain by silencing of TNF-α expression with lentiviral vectors targeting the dorsal root ganglion in mice

Neuropathic pain can be a debilitating condition. Many types of drugs that have been used to treat neuropathic pain have only limited efficacy. Recent studies indicate that pro-inflammatory mediators including tumor necrosis factor α (TNF-α) are involved in the pathogenesis of neuropathic pain. In the present study, we engineered a gene therapy strategy to relieve neuropathic pain by silencing TNF-α expression in the dorsal root ganglion (DRG) using lentiviral vectors expressing TNF short hairpin RNA1-4 (LV-TNF-shRNA1-4) in mice. First, based on its efficacy in silencing TNF-α in vitro, we selected shRNA3 to construct LV-TNF-shRNA3 for in vivo study. We used L5 spinal nerve transection (SNT) mice as a neuropathic pain model. These animals were found to display up-regulated mRNA expression of activating transcription factor 3 (ATF3) and neuropeptide Y (NPY), injury markers, and interleukin (IL)-6, an inflammatory cytokine in the ipsilateral L5 DRG. Injection of LV-TNF-shRNA3 onto the proximal transected site suppressed significantly the mRNA levels of ATF3, NPY and IL-6, reduced mechanical allodynia and neuronal cell death of DRG neurons. These results suggest that lentiviral-mediated silencing of TNF-α in DRG relieves neuropathic pain and reduces neuronal cell death, and may constitute a novel therapeutic option for neuropathic pain.

Haematopoietic cells produce BDNF and regulate appetite upon migration to the hypothalamus

Brain-derived neurotrophic factor (BDNF) suppresses food intake by acting on neurons in the hypothalamus. Here we show that BDNF-producing haematopoietic cells control appetite and energy balance by migrating to the hypothalamic paraventricular nucleus. These haematopoietic-derived paraventricular nucleus cells produce microglial markers and make direct contacts with neurons in response to feeding status. Mice with congenital BDNF deficiency, specifically in haematopoietic cells, develop hyperphagia, obesity and insulin resistance. These abnormalities are ameliorated by bone marrow transplantation with wild-type bone marrow cells. Furthermore, when injected into the third ventricle, wild-type bone marrow mononuclear cells home to the paraventricular nucleus and reverse the hyperphagia of BDNF-deficient mice. Our results suggest a novel mechanism of feeding control based on the production of BDNF by haematopoietic cells and highlight a potential new therapeutic route for the treatment of obesity.

Hyperglycemia induces abnormal gene expression in hematopoietic stem cells and their progeny in diabetic neuropathy

Diabetic peripheral neuropathy is a major chronic diabetic complication. We have previously shown that in type 1 diabetic streptozotocin‐treated mice, insulin‐ and TNF‐α co‐expressing bone marrow‐derived cells (BMDCs) induced by hyperglycemia travel to nerve tissues where they fuse with nerve cells, causing premature apoptosis and nerve dysfunction. Here we show that similar BMDCs also occur in type 2 diabetic high‐fat diet (HFD) mice. Furthermore, we found that hyperglycemia induces the co‐expression of insulin and TNF‐α in c‐kit+Sca‐1+lineage− (KSL) progenitor cells, which maintain the same expression pattern in the progeny, which in turn participates in the fusion with neurons when transferred to normoglycemic animals.

Clinical Phenotype and Segregation of Mitochondrial 3243A>G Mutation in 2 Pairs of Monozygotic Twins.

IMPORTANCE The regulatory factors explaining the wide spectrum of clinical phenotypes for mitochondrial 3243A>G mutation are not known. Crosstalk between nuclear genes and mitochondrial DNA might be one factor. OBSERVATIONS In this case series, we compared 2 pairs of male twins with the mitochondrial 3243 A>G mutation and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes syndrome with a female control patient. One pair of monozygotic twins presented with diabetes and deafness in their 30s, stroke-like episodes in their 40s, and cardiac events and death in their 50s. Another pair of twins presented with deafness and stroke-like episodes in their 20s. The degree of heteroplasmy of 3243A>G mutation in the various tissues and organs was similar in the first pair of twins compared with the control patient. CONCLUSIONS AND RELEVANCE The clinical phenotype and segregation of mitochondrial 3243A>G mutation was similar in monozygotic twins. The onset age and distribution of the symptoms might be regulated by nuclear genes. Our findings might help to predict the clinical course of the surviving twins and afford an opportunity for therapy before the onset of mitochondrial disease, especially for monozygotic twins caused by nuclear transfer with a small amount of nuclear-donor mitochondrial DNA.

Stem cell factor-activated bone marrow ameliorates amyotrophic lateral sclerosis by promoting protective microglial migration.

Amyotrophic lateral sclerosis (ALS) is a progressive disease associated with motor neuron death. Several experimental treatments, including cell therapy using hematopoietic or neuronal stem cells, have been tested in ALS animal models, but therapeutic benefits have been modest. Here we used a new therapeutic strategy, bone marrow transplantation (BMT) with stem cell factor (SCF)‐ or FMS‐like tyrosine kinase 3 (flt3)‐activated bone marrow (BM) cells for the treatment of hSOD1(G93A) transgenic mice. Motor function and survival showed greater improvement in the SCF group than in the group receiving BM cells that had not been activated (BMT alone group), although no improvement was shown in the flt3 group. In addition, larger numbers of BM‐derived cells that expressed the microglia marker Iba1 migrated to the spinal cords of recipient mice compared with the BMT‐alone group. Moreover, after SCF activation, but not flt3 activation or no activation, the migrating microglia expressed glutamate transporter‐1 (GLT‐1). In spinal cords in the SCF group, inflammatory cytokines tumor necrosis factor‐α and interleukin‐1β were suppressed and the neuroprotective molecule insulin‐like growth factor‐1 increased relative to nontreatment hSOD1(G93A) transgenic mice. Therefore, SCF activation changed the character of the migrating donor BM cells, which resulted in neuroprotective effects. These studies have identified SCF‐activated BM cells as a potential new therapeutic agent for the treatment of ALS.

Amyotrophic lateral sclerosis presenting with apraxia of speech.

Speech disturbance observed in patients with amyotrophic lateral sclerosis (ALS) usually arises from bulbar palsy. In those patients, atrophy and fasciculation of the tongue are observed and dysphagia might be complicated with speech disturbance in parallel. We herein describe an unusual woman with ALS whose initial symptom was apraxia of speech (AOS). A right-handed 66-year-old woman presented with a speech problem lasting for 6 months. She went to a dental clinic, departments of otolaryngology and neurosurgery at other hospitals, and a psychological clinic, but there was no abnormality detected in their examination. She was unable to speak or vocalize at all when she came to our hospital. There was no neurological disease in her family. Her parents were not consanguineous. She was alert and could walk by herself. There was no weakness of her face. Atrophy and fasciculation of the tongue was not observed. She did not complain of dysphagia. She could eat and drink anything without difficulty. There was no weakness in any of her limbs. Tendon reflexes were evoked normally. No pathological reflex was elicited. Sensory function and coordination were also normal. She could understand what we said. She could obey written commands. Although she could not speak, she could write what she wanted to say. Laryngoscopy and brain magnetic resonance imaging results were normal. Electromyography did not show any denervation potentials in muscles including the tongue. Single-photon emission computed tomography (SPECT) using I-N-isopropyl-P-iodoamphetamine revealed hypoperfusion at the middle and lower parts of the left precentral gyrus when analyzed with easy Z-score imaging system (Fig. 1) [1]. No objective examination indicated ALS at that time. One year after we had seen her for the first time, she came to our hospital requiring re-examination. There was no facial palsy, but forced laughing was observed. Atrophy and fasciculation of the tongue was seen. She did not complain of dysphagia. There was a weakness of her left lower limb. Tendon reflexes including jaw jerk were increased. Babinski sign was evoked bilaterally. The grammar of her sentences and morphology of her handwritten letters including kana (Japanese syllabograms) and kanji (Japanese ideograms) were almost correct. However, there was a phonological error in her handwriting (Fig. 2). Nerve conduction study showed decreased F wave frequency without conduction block. Electromyography revealed acute and chronic denervation potentials in her limbs. She was diagnosed as having ALS. In the next 3 years, she developed tetraplegia. Dysphagia, which developed slowly, required gastrostomy. Brain computed tomography 5 years after the onset showed atrophy of the medial temporal lobes but not the precentral gyri. Our patient’s initial symptom of speech problem was considered to be AOS. AOS is an impairment of motor planning and programming of speech articulation. Since K. Maeda (&) N. Ogawa R. Idehara Department of Neurology, National Hospital Organisation Higashi-ohmi General Medical Centre, 255 Gochi, Higashi-ohmi, Shiga 527-8505, Japan e-mail: maeda-kengo@shiga-hosp.jp

Temporal lobe epilepsy manifesting as alexia with agraphia for kanji

Alexia with agraphia results from lesions of the left angular gyrus or the left posteroinferior temporal lobe. In Japanese or Korean persons, lesions of the latter cause alexia with agraphia for ideograms. We describe a case of alexia with agraphia for kanji (Japanese ideograms) caused by temporal lobe epilepsy. After generalized convulsions, a 32-year-old man noticed that he had difficulty in reading and writing kanji, although he could read and write kana (Japanese syllabograms). His EEG showed frequent sharp waves on the left occipitotemporal region. MRI of the brain revealed a hyperintense lesion on the left hippocampus. Single-photon-emission computed tomography revealed marked hyperperfusion at the left hippocampus and the left posteroinferior temporal cortex. Antiepileptic drugs improved his alexia with agraphia for kanji. This is the first report describing alexia with agraphia for kanji caused by temporal lobe epilepsy.

Gene Therapy for Neuropathic Pain through siRNA-IRF5 Gene Delivery with Homing Peptides to Microglia

Astrocyte- and microglia-targeting peptides were identified and isolated using phage display technology. A series of procedures, including three cycles of both in vivo and in vitro biopanning, was performed separately in astrocytes and in M1 or M2 microglia, yielding 50–58 phage plaques in each cell type. Analyses of the sequences of this collection identified one candidate homing peptide targeting astrocytes (AS1[C-LNSSQPS-C]) and two candidate homing peptides targeting microglia (MG1[C-HHSSSAR-C] and MG2[C-NTGSPYE-C]). To determine peptide specificity for the target cell in vitro, each peptide was synthesized and introduced into the primary cultures of astrocytes or microglia. Those peptides could bind to the target cells and be selectively taken up by the corresponding cell, namely, astrocytes, M1 microglia, or M2 microglia. To confirm cell-specific gene delivery to M1 microglia, the complexes between peptide MG1 and siRNA-interferon regulatory factor 5 were prepared and intrathecally injected into a mouse model of neuropathic pain. The complexes successfully suppressed hyperalgesia with high efficiency in this neuropathic pain model. Here, we describe a novel gene therapy for the treatment neuropathic pain, which has a high potential to be of clinical relevance. This strategy will ensure the targeted delivery of therapeutic genes while minimizing side effects to non-target tissues or cells.