Myelodysplasia after clonal hematopoiesis with APOBEC3-mediated CYBB inactivation in retroviral gene therapy for X-CGD.

Stem cell gene therapy using the MFGS-gp91phox retroviral vector was performed on a 27-year-old patient with X-linked chronic granulomatous disease (X-CGD) in 2014. The patient's refractory infections were resolved, whereas the oxidase-positive neutrophils disappeared within 6 months. Thirty-two months after gene therapy, the patient developed myelodysplastic syndrome (MDS), and vector integration into the MECOM locus was identified in blast cells. The vector integration into MECOM was detectable in most myeloid cells at 12 months after gene therapy. However, the patient exhibited normal hematopoiesis until the onset of MDS, suggesting that MECOM transactivation contributed to clonal hematopoiesis, and the blast transformation likely arose after the acquisition of additional genetic lesions. In whole-genome sequencing, the biallelic loss of the WT1 tumor suppressor gene, which occurred immediately before tumorigenesis, was identified as a potential candidate genetic alteration. The provirus CYBB cDNA in the blasts contained 108 G-to-A mutations exclusively in the coding strand, suggesting the occurrence of APOBEC3-mediated hypermutations during the transduction of CD34-positive cells. A hypermutation-mediated loss of oxidase activity may have facilitated the survival and proliferation of the clone with MECOM transactivation. Our data provide valuable insights into the complex mechanisms underlying the development of leukemia in X-CGD gene therapy.

Effects of Thoracic Spine Self-mobilization on Patients with Low Back Pain and Lumbar Hypermobility: A Randomized Controlled Trial.

Objectives: This study used magnetic resonance imaging (MRI) to investigate the effects of thoracic spine self-mobilization on patients with low back pain (LBP) and lumbar hypermobility. Methods: Twenty-four patients (15 men, 9 women) with LBP were randomly allocated to a thoracic spine self-mobilization group or sham group. The thoracic spine self-mobilization group performed thoracic spine active flexion and extension activities using two tennis balls fixed with athletic tape. Outcome measures were collected pre-intervention and after 4 weeks and included the Visual Analog Scale (VAS) for pain, the Oswestry Disability Index, lumbar rotation angle measured using MRI taken in the lateral position with 45° of trunk rotation, thoracolumbar rotation range of motion (ROM) in the sitting position, and stiffness of the erector spinae muscles. The effects of the intervention were analyzed using two-way repeated-measures analysis of variance (ANOVA), followed by multiple comparisons. The significance level was set at 5%. Results: The results of the two-way repeated measures ANOVA indicated that the main effect of the group was significant (P<0.05) for VAS, the sum of the lumbar rotation angle, and the thoracolumbar rotation ROM. A significant group-by-time interaction was found for the sum of lumbar rotation angles. The results of the multiple comparison tests for VAS, sum of the lumbar rotation angle from L1 to S1, and thoracolumbar rotation ROM were significantly different after 4 weeks. Conclusions: This study revealed a decrease in lumbar segmentation after thoracic spine mobilization. Thoracic spine mobilization may be effective in patients with LBP and hypermobility.

Peripheral immune system modulates Purkinje cell degeneration in Niemann-Pick disease type C1.

Niemann-Pick disease type C1 (NPC1) is a fatal lysosomal storage disorder characterized by progressive neuronal degeneration. Its key pathogenic events remain largely unknown. We have, herein, found that neonatal BM-derived cell transplantation can ameliorate Purkinje cell degeneration in NPC1 mice. We subsequently addressed the impact of the peripheral immune system on the neuropathogenesis observed in NPC1 mice. The depletion of mature lymphocytes promoted NPC1 phenotypes, thereby suggesting a neuroprotective effect of lymphocytes. Moreover, the peripheral infusion of CD4-positive cells (specifically, of regulatory T cells) from normal healthy donor ameliorated the cerebellar ataxic phenotype and enhanced the survival of Purkinje cells. Conversely, the depletion of regulatory T cells enhanced the onset of the neurological phenotype. On the other hand, circulating inflammatory monocytes were found to be involved in the progression of Purkinje cell degeneration, whereas the depletion of resident microglia had little effect. Our findings reveal a novel role of the adaptive and the innate immune systems in NPC1 neuropathology.

Analysis of cervical and upper thoracic spinal segmental rotation angles during end-range neck rotation: Comparison with and without neck pain.

BACKGROUND: Neck pain is a common manifestation of musculoskeletal disorders of the cervical and thoracic spine. Manual therapy interventions to the thoracic spine are recommended for treating patients with several types of neck pain. However, only a few studies have investigated the thoracic spine mobility associated with neck movement. OBJECTIVES: Compare cervical and upper thoracic rotation angles in subjects with and without neck pain. METHODS: The subjects included nine individuals who experienced neck pain (pain, Group P) and 11 who did not (non-pain, Group N). The rotation angle was measured using MRI. The imaging limb position was at 90% of the maximum neck rotation. The MR images were analyzed using image analysis software to calculate the rotation angle of C1 to Th3. The rotation angle of the segment was then calculated by subtracting the rotation angle corresponding to the lower vertebra from that corresponding to the upper vertebra. The total rotation of each segment was calculated as the sum of the right and left rotation angle. Then, the segmental rotation angles were compared between groups. RESULTS/FINDINGS: The rotation angles of C3-C4, C7-Th1, and Th1-Th2 were significantly smaller in Group P than in Group N, and C5-C6 and C6-C7 were significantly larger in Group P than in Group N. There was no statistical difference in rotational angle at all other spinal levels measured. CONCLUSIONS: The results of this study indicate subjects with neck pain had hypermobility of the lower cervical spine and hypomobility of the cervico-thoracic junction and upper thoracic spine compared with subjects without neck pain. These results add to current understanding of biomechanical factors that may be related to neck pain.

Nonconditioned ADA-SCID gene therapy reveals ADA requirement in the hematopoietic system and clonal dominance of vector-marked clones.

Two patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency (ADA-SCID) received stem cell-based gene therapy (SCGT) using GCsapM-ADA retroviral vectors without preconditioning in 2003 and 2004. The first patient (Pt1) was treated at 4.7 years old, and the second patient (Pt2), who had previously received T cell gene therapy (TCGT), was treated at 13 years old. More than 10 years after SCGT, T cells showed a higher vector copy number (VCN) than other lineages. Moreover, the VCN increased with differentiation toward memory T and B cells. The distribution of vector-marked cells reflected variable levels of ADA requirements in hematopoietic subpopulations. Although neither patient developed leukemia, clonal expansion of SCGT-derived clones was observed in both patients. The use of retroviral vectors yielded clonal dominance of vector-marked clones, irrespective of the lack of leukemic changes. Vector integration sites common to all hematopoietic lineages suggested the engraftment of gene-marked progenitors in Pt1, who showed severe osteoblast (OB) insufficiency compared to Pt2, which might cause a reduction in the stem/progenitor cells in the bone marrow (BM). The impaired BM microenvironment due to metabolic abnormalities may create space for the engraftment of vector-marked cells in ADA-SCID, despite the lack of preconditioning.

Modified central extracorporeal membrane oxygenation for distended left ventricle.

A 38-year-old man who was resuscitated from ventricular fibrillation was diagnosed with acute aortic dissection complicated by coronary malperfusion. He underwent total aortic arch replacement and coronary artery bypass grafting to the left anterior descending coronary artery. Due to low cardiac output syndrome from cardiac ischemia, central extracorporeal membrane oxygenator (ECMO) was established with aortic cannulation from the side branch of the implanted prosthetic graft and venous drainage from the femoral vein. Ventricular venting was added from the right upper pulmonary vein for the distended left ventricle. ECMO was weaned off on postoperative Day 4. The patient is back on his normal daily life for more than 1 year after the surgery.

Minimized perfusion circuit for acute type A aortic dissection surgery.

A minimized perfusion circuit (MPC) may reduce transfusion requirement and inflammatory response. Its use, however, has not been standardized for complicated cardiovascular surgery. We assessed outcomes of surgery for acute type A aortic dissection (ATAAD) performed with a MPC under circulatory arrest. The study involved 706 patients treated surgically for ATAAD (by hemiarch repair [n = 571] or total arch repair [n = 135]). Total arch repair was performed using selective antegrade cerebral perfusion. Our MPC, a semi-closed bypass system, incorporating a completely closed circuit and a level-sensing reservoir in the venous circuit, was used. Clinical variables, transfusion volume, and outcomes were investigated in patients who underwent hemiarch repair or total arch repair. The overall incidences of shock, organ ischemia, and coagulopathy (prothrombin time-international normalized ratio >1.5) were 26%, 35%, and 8%, respectively. Mean extracorporeal circulation (ECC) time was 149 minutes for the hemiarch repair group and 241 minutes for the total arch repair group, respectively. No patient required conversion to conventional ECC, and there were no complications related to the use of the MPC. The need for transfusion (98% vs. 91%, P = .017) and median transfusion volume (1970 vs. 1680 mL, P = .002) was increased in the total arch repair group. Neither in-hospital mortality (total arch; 12% vs. hemiarch; 7%, P = .11) nor 10-year survival (74.4% vs. 68.4%, P = .79) differed significantly. Outcomes of surgery for ATAAD performed with the MPC were acceptable. The possibility of transfusion and transfusion volume remains high during such surgery, despite the use of the MPC.

Promotion of mitochondrial biogenesis by necdin protects neurons against mitochondrial insults.

Neurons rely heavily on mitochondria for their function and survival. Mitochondrial dysfunction contributes to the pathogenesis of neurodegenerative diseases such as Parkinson's disease. PGC-1α is a master regulator of mitochondrial biogenesis and function. Here we identify necdin as a potent PGC-1α stabilizer that promotes mitochondrial biogenesis via PGC-1α in mammalian neurons. Expression of genes encoding mitochondria-specific proteins decreases significantly in necdin-null cortical neurons, where mitochondrial function and expression of the PGC-1α protein are reduced. Necdin strongly stabilizes PGC-1α by inhibiting its ubiquitin-dependent degradation. Forced expression of necdin enhances mitochondrial function in primary cortical neurons and human SH-SY5Y neuroblastoma cells to prevent mitochondrial respiratory chain inhibitor-induced degeneration. Moreover, overexpression of necdin in the substantia nigra in vivo of adult mice protects dopaminergic neurons against degeneration in experimental Parkinson's disease. These data reveal that necdin promotes mitochondrial biogenesis through stabilization of endogenous PGC-1α to exert neuroprotection against mitochondrial insults.

A novel histone deacetylase 1 and 2 isoform-specific inhibitor alleviates experimental Parkinson's disease.

With increased histone deacetylase (HDAC) activity and histone hypoacetylation being implicated in neurodegeneration, HDAC inhibitors have been reported to have considerable therapeutic potential. Yet, existing inhibitors lack specificity and may show substantial adverse effect. In this study, we identified a novel HDAC1/2 isoform-specific inhibitor, K560, with protective effects against 1-methyl-4-phenylpyridinium (MPP(+))- and/or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neuronal death in both in vitro and in vivo Parkinson's disease model. K560 attenuated cell death induced by MPP(+) in differentiated SH-SY5Y cells through the sustained expression of an antiapoptotic protein, X-linked inhibitor of apoptosis (XIAP). Inhibition of XIAP expression by locked nucleic acid antisense oligonucleotides abolished the protective effect of K560. Inactivation of mitogen-activated protein kinase cascades, reduced p53 phosphorylation, and down-regulation of p53-upregulated modulator of apoptosis on K560 treatment were also observed. Furthermore, pre- and post-oral administration of K560 to mice prevented MPTP-induced loss of dopaminergic neurons in substantia nigra, suggesting that selective inhibition of HDAC1 and HDAC2 by K560 may pave the way to new strategies for Parkinson's disease treatment.

Deficiency of Calcium-Independent Phospholipase A2 Beta Induces Brain Iron Accumulation through Upregulation of Divalent Metal Transporter 1.

Mutations in PLA2G6 have been proposed to be the cause of neurodegeneration with brain iron accumulation type 2. The present study aimed to clarify the mechanism underlying brain iron accumulation during the deficiency of calcium-independent phospholipase A2 beta (iPLA2ß), which is encoded by the PLA2G6 gene. Perl's staining with diaminobenzidine enhancement was used to visualize brain iron accumulation. Western blotting was used to investigate the expression of molecules involved in iron homeostasis, including divalent metal transporter 1 (DMT1) and iron regulatory proteins (IRP1 and 2), in the brains of iPLA2ß-knockout (KO) mice as well as in PLA2G6-knockdown (KD) SH-SY5Y human neuroblastoma cells. Furthermore, mitochondrial functions such as ATP production were examined. We have discovered for the first time that marked iron deposition was observed in the brains of iPLA2ß-KO mice since the early clinical stages. DMT1 and IRP2 were markedly upregulated in all examined brain regions of aged iPLA2ß-KO mice compared to age-matched wild-type control mice. Moreover, peroxidized lipids were increased in the brains of iPLA2ß-KO mice. DMT1 and IRPs were significantly upregulated in PLA2G6-KD cells compared with cells treated with negative control siRNA. Degeneration of the mitochondrial inner membrane and decrease of ATP production were observed in PLA2G6-KD cells. These results suggest that the genetic ablation of iPLA2ß increased iron uptake in the brain through the activation of IRP2 and upregulation of DMT1, which may be associated with mitochondrial dysfunction.

Functional Domains of ZFP809 Essential for Nuclear Localization and Gene Silencing.

Zinc finger protein 809 (ZFP809) is a member of the Kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family, and is highly expressed in mouse immature cells. ZFP809 is known to inhibit the expression of transduced genes driven by Moloney murine leukemia virus (MoMLV)-typed retroviral vectors by binding to the primer binding site (PBS) located downstream of the MLV-long terminal repeat (LTR) of the vectors and recruiting protein complexes that introduce epigenetic silencing marks such as histone modifications and DNA methylation at the MLV-LTR. However, it remains undetermined what domains of ZFP809 among the KRAB domain at N-terminus and the seven zinc fingers are critical for gene silencing. In this study, we assessed subcellular localization, gene silencing ability, and binding ability to the PBS of a series of truncated and mutated ZFP809 proteins. We revealed the essential role of the KRAB A box for all functions assessed, together with the accessory roles of a subset of zinc fingers. Our data also suggest that interaction between KAP1 and the KRAB A box of ZFP809 is critical in KAP1-dependent control of gene silencing for ZFP809 targets.

The promises of stem cells: stem cell therapy for movement disorders.

Despite the multitude of intensive research, the exact pathophysiological mechanisms underlying movement disorders including Parkinson's disease, multiple system atrophy and Huntington's disease remain more or less elusive. Treatments to halt these disease progressions are currently unavailable. With the recent induced pluripotent stem cells breakthrough and accomplishment, stem cell research, as the vast majority of scientists agree, holds great promise for relieving and treating debilitating movement disorders. As stem cells are the precursors of all cells in the human body, an understanding of the molecular mechanisms that govern how they develop and work would provide us many fundamental insights into human biology of health and disease. Moreover, stem-cell-derived neurons may be a renewable source of replacement cells for damaged neurons in movement disorders. While stem cells show potential for regenerative medicine, their use as tools for research and drug testing is thought to have more immediate impact. The use of stem-cell-based drug screening technology could be a big boost in drug discovery for these movement disorders. Particular attention should also be given to the involvement of neural stem cells in adult neurogenesis so as to encourage its development as a therapeutic option.

Neurodegenerative changes initiated by presynaptic dysfunction.

α-Synucleinopathies are a subgroup of neurodegenerative diseases including dementia with Lewy bodies (DLB) and Parkinson's disease (PD). Pathologically, these disorders can be characterized by the presence of intraneuronal aggregates composed mainly of α-synuclein (αSyn), which are called Lewy bodies and Lewy neurites. Recent report showed that more than 90% of αSyn aggregates are present in the form of very small deposits in presynaptic terminals of the affected neurons in DLB. However, the mechanisms responsible for presynaptic accumulation of abnormal αSyn remain unclear. In this article, we review recent findings on the involvement of presynaptic dysfunction in the initiation of neuronal dysfunctional changes. This review highlights that the presynaptic failure can be a potential trigger of the dying-back neuronal death in neurodegenerative diseases.

α-Synuclein and neuronal cell death.

Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting ~1 % of people over the age of 65. Neuropathological hallmarks of PD are prominent loss of dopaminergic (DA) neurons in the substantia nigra and formation of intraneuronal protein inclusions termed Lewy bodies, composed mainly of α-synuclein (αSyn). Missense mutations in αSyn gene giving rise to production of degradation-resistant mutant proteins or multiplication of wild-type αSyn gene allele can cause rare inherited forms of PD. Therefore, the existence of abnormally high amount of αSyn protein is considered responsible for the DA neuronal death in PD. Normally, αSyn protein localizes to presynaptic terminals of neuronal cells, regulating the neurotransmitter release through the modulation of assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex. On the other hand, of note, pathological examinations on the recipient patients of fetal nigral transplants provided a prion-like cell-to-cell transmission hypothesis for abnormal αSyn. The extracellular αSyn fibrils can internalize to the cells and enhance intracellular formation of protein inclusions, thereby reducing cell viability. These findings suggest that effective removal of abnormal species of αSyn in the extracellular space as well as intracellular compartments can be of therapeutic relevance. In this review, we will focus on αSyn-triggered neuronal cell death and provide possible disease-modifying therapies targeting abnormally accumulating αSyn.

Accumulation of α-synuclein triggered by presynaptic dysfunction.

Pathological examination of dementia with Lewy bodies patients identified the presence of abnormal α-synuclein (αSyn) aggregates in the presynaptic terminals. αSyn is involved in the regulation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Importantly, αSyn-transgenic mouse and postmortem examination of patients with Parkinson's disease have demonstrated the abnormal distribution of SNARE protein in presynaptic terminals. In this study, we investigated the effects of SNARE dysfunction on endogenous αSyn using Snap25(S187A/S187A) mutant mice. These mice have homozygous knock-in gene encoding unphosphorylatable S187A-substituted synaptosomal-associated protein of 25 kDa (SNAP-25). The mice displayed a significant age-dependent change in the distribution of αSyn and its Ser(129)-phosphorylated form in abnormally hypertrophied glutamatergic nerve terminals in the striatum. Electron-microscopic analysis revealed the abnormally condensed synaptic vesicles with concomitant mislocalization of αSyn protein to the periactive zone in the glutamatergic nerve terminals. However, the Snap25(S187A/S187A) mutant mouse harbored no abnormalities in the nigrostriatal dopaminergic neurons. Our present results suggest that SNARE dysfunction is the initial trigger of mislocalization and accumulation of αSyn, and probably is an important pathomechanism of α-synucleinopathies.

Iron accumulation in Parkinson's disease.

Although the exact cause of Parkinson's disease (PD) is still unknown, recent interest has been focused on the role of iron in the nigral cell death in PD. Several studies have shown that a selective and significant elevation in iron occurs in the substantia nigra of patients with PD. However, the mechanisms involved in iron accumulation also remain unclear. In this article, we describe recent findings regarding the mechanisms and potential toxic effects of iron accumulation in hereditary and sporadic PD and animal models of PD, including our genetic mouse model of PD. The review provides an opportunity to revisit the possible roles of iron accumulation in the pathogenic cascade(s) of PD.

Adeno-associated viral vector-mediated gene transduction in mesencephalic slice culture.

Adeno-associated viral (AAV) vector is a non-pathogenic vehicle that is suitable for the delivery of foreign genes into non-dividing neuronal cells. This vector has been utilized for in vivo neurological research and in clinical trials of gene therapy for neurodegenerative disorders. Viral vector-mediated gene delivery has the limitation that progressive changes in cellular phenotype cannot be monitored in living animals. To visualize living neurons transduced with foreign genes in vitro, we used cultured mesencephalic tissue harboring living dopaminergic (DA) neurons and examined cellular tropism of serotype-1 and serotype-2 AAV vectors in a culture system. The viability of DA neurons was evaluated using transgenic mice carrying enhanced green fluorescent protein under the control of the rat tyrosine hydroxylase (TH) promoter, which enables the visualization of living DA cells in the substantia nigra. Apoptosis of a subset of neuronal cells was noted within one day of culture. After 7 days, the serotype-1 AAV vector had successfully delivered the foreign gene into neurons and astrocytes, and serotype-2 AAV vector was able to transduce TH-positive DA neurons efficiently. Our method should be useful for in vitro investigations of pathological changes in DA neurons following transduction with foreign genes.

Parkin-mediated protection of dopaminergic neurons in a chronic MPTP-minipump mouse model of Parkinson disease.

Loss-of-function mutations in the ubiquitin ligase parkin are the major cause of recessively inherited early-onset Parkinson disease (PD). Impairment of parkin activity caused by nitrosative or dopamine-related modifications may also be responsible for the loss of dopaminergic (DA) neurons in sporadic PD. Previous studies have shown that viral vector-mediated delivery of parkin prevented DA neurodegeneration in several animal models, but little is known about the neuroprotective actions of parkin in vivo. Here, we investigated mechanisms of neuroprotection of overexpressed parkin in a modified long-term mouse model of PD using osmotic minipump administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Recombinant adeno-associated viral vector-mediated intranigral delivery of parkin prevented motor deficits and DA cell loss in the mice. Ser129-phosphorylated α-synuclein-immunoreactive cells were increased in the substantia nigra of parkin-treated mice. Moreover, delivery of parkin alleviated the MPTP-induced decrease of the active phosphorylated form of Akt. On the other hand, upregulation of p53 and mitochondrial alterations induced by chronic MPTP administration were barely suppressed by parkin. These results suggest that the neuroprotective actions of parkin may be impaired in severe PD.

Use of growth factors for the treatment of Parkinson's disease.

Neurotrophic factors are a subset of growth factors that promote survival, differentiation and maintenance of neuronal cells in the developing and adult nervous systems. These factors can potentially be a vital tool for the treatment of progressive neurodegenerative disorders, including Parkinson's disease (PD). Impairments of initiation and control of movements caused by significant degeneration of the nigrostriatal dopaminergic neurons in PD can be targeted by intracerebral infusion of recombinant protein, cell-based ex vivo therapy or viral vector-mediated in vivo gene therapy using the neurotrophic factors. Since the finding that the glial cell line-derived neurotrophic factor might promote survival and regeneration of dopaminergic neurons in experimental models and recent clinical trials, the use of neurotrophic factors has been considered central in novel therapeutic strategies that slow or reverse the deleterious symptoms in advanced PD. This article summarizes a series of neurotrophic factors that provide neuroprotection or restoration for dopaminergic neurons in PD, and the potential clinical availability of this approach for the treatment of patients with PD.

Striatal overexpression of DeltaFosB reproduces chronic levodopa-induced involuntary movements.

Long-term dopamine replacement therapy in Parkinson's disease leads to the development of disabling involuntary movements named dyskinesias that are related to adaptive changes in striatal signaling pathways. The chronic transcription factor DeltaFosB, which is overexpressed in striatal neurons after chronic dopaminergic drug exposure, is suspected to mediate these adaptive changes. Here, we sought to demonstrate the ability of DeltaFosB to lead directly to the abnormal motor responses associated with chronic dopaminergic therapy. Using rAAV (recombinant adenoassociated virus) viral vectors, high levels of DeltaFosB expression were induced in the striatum of dopamine-denervated rats naive of chronic drug administration. Transgenic DeltaFosB overexpression reproduced the entire spectrum of altered motor behaviors in response to acute levodopa tests, including different types of abnormal involuntary movements and hypersensitivity of rotational responses that are typically associated with chronic levodopa treatment. JunD, the usual protein partner of DeltaFosB binding to AP-1 (activator protein-1) sites of genes, remained unchanged in rats with high DeltaFosB expression induced by viral vectors. These findings demonstrate that the increase of striatal DeltaFosB in the evolution of chronically treated Parkinson's disease may be a trigger for the development of abnormal responsiveness to dopamine and the emergence of involuntary movements.