Effects of local reduction of endogenous α-synuclein using antisense oligonucleotides on the fibril-induced propagation of pathology through the neural network in wild-type mice.

In Parkinson's disease and other synucleinopathies, fibrillar forms of α-synuclein (aSyn) are hypothesized to structurally convert and pathologize endogenous aSyn, which then propagates through the neural connections, forming Lewy pathologies and ultimately causing neurodegeneration. Inoculation of mouse-derived aSyn preformed fibrils (PFFs) into the unilateral striatum of wild-type mice causes widespread aSyn pathologies in the brain through the neural network. Here, we used the local injection of antisense oligonucleotides (ASOs) against Snca mRNA to confine the area of endogenous aSyn protein reduction and not to affect the PFFs properties in this model. We then varied the timing and location of ASOs injection to examine their impact on the initiation and propagation of aSyn pathologies in the whole brain and the therapeutic effect using abnormally-phosphorylated aSyn (pSyn) as an indicator. By injecting ASOs before or 0-14 days after the PFFs were inoculated into the same site in the left striatum, the reduction in endogenous aSyn in the striatum leads to the prevention and inhibition of the regional spread of pSyn pathologies to the whole brain including the contralateral right hemisphere. ASO post-injection inhibited extension from neuritic pathologies to somatic ones. Moreover, injection of ASOs into the right striatum prevented the remote regional spread of pSyn pathologies from the left striatum where PFFs were inoculated and no ASO treatment was conducted. This indicated that the reduction in endogenous aSyn protein levels at the propagation destination site can attenuate pSyn pathologies, even if those at the propagation initiation site are not inhibited, which is consistent with the original concept of prion-like propagation that endogenous aSyn is indispensable for this regional spread. Our results demonstrate the importance of recruiting endogenous aSyn in this neural network propagation model and indicate a possible potential for ASO treatment in synucleinopathies.

Clinical course of pathologically confirmed corticobasal degeneration and corticobasal syndrome.

The clinical presentation of corticobasal degeneration is diverse, while the background pathology of corticobasal syndrome is also heterogeneous. Therefore, predicting the pathological background of corticobasal syndrome is extremely difficult. Herein, we investigated the clinical findings and course in patients with pathologically, genetically and biochemically verified corticobasal degeneration and corticobasal syndrome with background pathology to determine findings suggestive of background disorder. Thirty-two patients were identified as having corticobasal degeneration. The median intervals from the initial symptoms to the onset of key milestones were as follows: gait disturbance, 0.0 year; behavioural changes, 1.0 year; falls, 2.0 years; cognitive impairment, 2.0 years; speech impairment, 2.5 years; supranuclear gaze palsy, 3.0 years; urinary incontinence, 3.0 years; and dysphagia, 5.0 years. The median survival time was 7.0 years; 50% of corticobasal degeneration was diagnosed as corticobasal degeneration/corticobasal syndrome at the final presentation. Background pathologies of corticobasal syndrome (n = 48) included corticobasal degeneration (33.3%), progressive supranuclear palsy (29.2%) and Alzheimer's disease (12.5%). The common course of corticobasal syndrome was initial gait disturbance and early fall. In addition, corticobasal degeneration-corticobasal syndrome manifested behavioural change (2.5 years) and cognitive impairment (3.0 years), as the patient with progressive supranuclear palsy-corticobasal syndrome developed speech impairment (1.0 years) and supranuclear gaze palsy (6.0 years). The Alzheimer's disease-corticobasal syndrome patients showed cognitive impairment (1.0 years). The frequency of frozen gait at onset was higher in the corticobasal degeneration-corticobasal syndrome group than in the progressive supranuclear palsy-corticobasal syndrome group [P = 0.005, odds ratio (95% confidence interval): 31.67 (1.46-685.34)]. Dysarthria at presentation was higher in progressive supranuclear palsy-corticobasal syndrome than in corticobasal degeneration-corticobasal syndrome [P = 0.047, 6.75 (1.16-39.20)]. Pyramidal sign at presentation and personality change during the entire course were higher in Alzheimer's disease-corticobasal syndrome than in progressive supranuclear palsy-corticobasal syndrome [P = 0.011, 27.44 (1.25-601.61), and P = 0.013, 40.00 (1.98-807.14), respectively]. In corticobasal syndrome, decision tree analysis revealed that 'freezing at onset' or 'no dysarthria at presentation and age at onset under 66 years in the case without freezing at onset' predicted corticobasal degeneration pathology with a sensitivity of 81.3% and specificity of 84.4%. 'Dysarthria at presentation and age at onset over 61 years' suggested progressive supranuclear palsy pathology, and 'pyramidal sign at presentation and personality change during the entire course' implied Alzheimer's disease pathology. In conclusion, frozen gait at onset, dysarthria, personality change and pyramidal signs may be useful clinical signs for predicting background pathologies in corticobasal syndrome.

Recurrent Lymphocytic Hypophysitis Presenting as Internal Carotid Artery Stenosis and Oculomotor Nerve Palsy.

A 75-year-old woman presented with nausea and vomiting. Magnetic resonance imaging (MRI) revealed that she had a pituitary mass. A biopsy revealed lymphocytic hypophysitis (LYH). Symptoms were improved by hormone replacement therapy. Although she was asymptomatic, follow-up MRI revealed an increase in the size of the mass. Intravenous methylprednisolone (IVMP) reduced the size of the mass; however, right ophthalmalgia and oculomotor nerve palsy developed. MRI showed that the pituitary mass had enlarged to the right oculomotor nerve in the cavernous sinus and to the right internal carotid artery (ICA), causing stenosis of the ICA. After IVMP administration, the symptoms dramatically improved, but ICA stenosis persisted.

Parallel Appearance of Polyglutamine and Transactivation-Responsive DNA-Binding Protein 43 and Their Complementary Subcellular Localization in Brains of Patients With Spinocerebellar Ataxia Type 2.

Spinocerebellar ataxia type 2 (SCA2) is caused by mutations in the ATXN2 gene in which toxic effects are triggered by expanded polyglutamine repeats within ataxin-2. SCA2 is accompanied by motor neuron degeneration as occurs in amyotrophic lateral sclerosis (ALS). We investigated the distribution patterns of ataxin-2 and transactivation-responsive DNA-binding protein 43 (TDP-43), a major disease-related protein in ALS, in the CNS of 3 SCA2 patients. Phosphorylated TDP-43 (pTDP-43)-positive lesions were widely distributed throughout the CNS and generally overlapped with 1C2 (expanded polyglutamine)-immunoreactive lesions. This distribution pattern is different from the pattern in limbic-predominant age-related TDP-43 encephalopathy. In SCA2, double immunostaining of TDP-43 and 1C2 in motor neurons revealed 3 staining patterns: cytoplasmic 1C2 and nuclear TDP-43, nucleocytoplasmic 1C2 and nuclear TDP-43, and nuclear 1C2 and cytoplasmic TDP-43, which reflect the early, active, and final stages of pathological change, respectively. The translocation of TDP-43 from the nucleus to the cytoplasm along with the translocation of 1C2 in the opposite direction indicates that nuclear accumulation of the disease-specific protein ataxin-2 affects the intracellular dynamics of TDP-43. Such a close interrelationship between mutant ataxin-2 and TDP-43 in the cell might account for the similarity of their distribution in the CNS of patients with SCA2.

Neuronal internalization of immunoglobulin G injected into the mouse brain by a novel absorption strategy to avoid unwanted interaction with immune complex using centrifugal filtration.

Monoclonal immunoglobulin-G (IgG) antibodies are now emerging as therapeutic tools to tackle various disorders, including those affecting the brain. However, little is known about how these IgG molecules behave in the brain. To better understand the potential behavior of IgG molecules in the brain, here we established a specific protocol to immunolocalize rat IgG injected into mouse striatum with an anti-rat IgG antibody. Using double immunolabeling, IgG-like immunoreactivity (IR) was mainly found in neurons but scarcely observed in glia 1 h after intrastriatal injection of IgG, whereas some surrounding glia contained IgG-like IR 24 h after injection. However, preabsorption with a large excess of rat IgG to confirm the authenticity of this labeling failed to eliminate this neuronal IgG-like IR but rather exhibited nuclear staining in glial cells. Because this unexpected nuclear staining escalated with increasing amount of absorbing IgG, we postulated that this nuclear staining is due to formation of immune complex IgG-anti-IgG, which can be removed by centrifugal filtration. As expected, this nuclear staining in glial cells was eliminated after centrifugal filtration of the IgG/anti-IgG mixture, and authentic IgG-like IR was chiefly detected in the cytoplasm of neurons around the injection channel. This study is the first demonstration of neuronal redistribution of injected IgG in the mouse brain. Neuronal internalization of exogenous IgG may be advantageous especially when the therapeutic targets of monoclonal IgG are intraneuronal such as neurofibrillary tangles or Lewy bodies.

Parallel gold enhancement of quantum dots 565/655 for double-labelling correlative light and electron microscopy on human autopsied samples.

Cadmium selenide quantum dots (QDs) are fluorescent and electron-dense nanoparticles. When used as reporter of immunolabeling, this dual visibility is essential for direct comparison of its fluorescent signals on light microscopy (LM) and their ultrastructrual counterparts on electron microscopy (EM) as correlative light and electron microscopy (CLEM). To facilitate EM recognition, QDs on EM grid were gold enhanced, which increased their size and electron density. On histological sections as well, gold-enhanced QDs, used as a reporter of immunolabeling, were easily recognized on EM. Because target structures are visible on bright field microscopy, gold enhancement facilitated trimming the target structures into final EM sections. Furthermore, gold enhancement of rod-shaped QD655 on EM grid was accentuated on their tips while spherical QD565 was gold-enhanced as sphere in contrast. This EM distinction was evident on histological sections where QD565 (green fluorescence) and QD655 (red fluorescence) were used as a reporter pair for double immunolabeling. Double-labeled immuno-fluorescent images, initially captured before EM processing, are now compared with their respective immuno EM counterparts. Specific labeling of each epitope was corroborated by mutual comparison between LM and EM. Although fluoronanogold may be a candidate reporter partner with QDs for gold-enhanced, double-labeling CLEM, its limited penetration into fixed tissue hampers universal use for thick histological sections. Gold-enhancement of QD immunolabeling, now expanded to double-labeling CLEM for human brain samples, will pave the way to translate molecular events into ultrastructural morphopathogenesis in situ.

Serial ultrathin sections to identify ultrastructural localization of GLUT1 molecules in vesicles in brain endothelial cells-correlative light and electron microscopy in depth.

Precise immunolocalization of molecules in relation to ultrastructural features is challenging, especially when the target is small and not frequent enough to be included in tiny ultrathin sections randomly selected for electron microscopy (EM). Glucose transporter 1 (GLUT1) is in charge of transporting glucose across brain capillary endothelial cells (BCECs). Paraformaldehyde-fixed floating sections (50 µm thick) of mouse brain were immunolabeled with anti-GLUT1 antibody and visualized with fluoronanogold. Fluorescent images encompassing the entire hemisphere were tiled to enable selection of GLUT1-positive BCECs suitable for subsequent EM and landmark placement with laser microdissection to guide trimming. Sections were then fixed with glutaraldehyde, gold enhanced to intensify the labeling and fixed with osmium tetroxide to facilitate ultrastructural recognition. Even though a region that contained target BCECs was successfully trimmed in the resin block, it was only after observation of serial ultrathin sections that GLUT1 signals in coated vesicles on the same cross section corresponding to the cross section preidentified by confocal laser microscope. This is the first ultrastructural demonstration of GLUT1 molecules in coated vesicles, which may well explain its functional relevance to transport glucose across BCECs. Successful ultrastructural localization of molecules in relation to well-preserved target structure in native tissue samples, as achieved in this study, will pave the way to understand the functional relevance of molecules and their relation to ultrastructural details.

Decreased plasma ghrelin in male ALS patients is associated with poor prognosis.

Physiological changes including altered nutritional status influence disease progression and survival in patients with amyotrophic lateral sclerosis (ALS). Ghrelin affects the nutritional status by regulating appetite and energy expenditure, and also has neuroprotective effects. To investigate the association between ghrelin and ALS prognosis, we analyzed plasma acylated-ghrelin levels in 33 patients with ALS. Compared among ALS patients, male had lower plasma ghrelin levels than female, although disease specificity is unknown. ALS patients, especially male ALS patients, with low plasma ghrelin levels (<15 fmol/mL) had significantly shorter post-examination survival times than those with high plasma ghrelin levels (≥15 fmol/mL). Univariate and multivariate analyses revealed a significant effect of ghrelin levels on post-examination survival. Immunohistochemical study of autopsied stomach samples from 8 of 33 patients revealed that the population of ghrelin-positive cells tended to be reduced in the low-plasma ghrelin group than in the high-plasma ghrelin group. Our findings suggest that ghrelin levels are an independent predictor of survival in ALS, especially male ALS patients, and the ghrelin-positive cells may decrease in ALS with low plasma ghrelin. Thus, reduced ghrelin secretion may be associated with poor prognosis among patients with ALS.

Neuropathology of SCA34 showing widespread oligodendroglial pathology with vacuolar white matter degeneration: a case study.

Spinocerebellar ataxia type 34 (SCA34) is an autosomal dominant inherited ataxia due to mutations in ELOVL4, which encodes one of the very long-chain fatty acid elongases. SCA38, another spinocerebellar ataxia, is caused by mutations in ELOVL5, a gene encoding another elongase. However, there have been no previous studies describing the neuropathology of either SCA34 or 38. This report describes the neuropathological findings of an 83-year-old man with SCA34 carrying a pathological ELOVL4 mutation (NM_022726, c.736T>G, p.W246G). Macroscopic findings include atrophies in the pontine base, cerebellum, and cerebral cortices. Microscopically, marked neuronal and pontocerebellar fiber loss was observed in the pontine base. In addition, in the pontine base, accumulation of CD68-positive macrophages laden with periodic acid-Schiff (PAS)-positive material was observed. Many vacuolar lesions were found in the white matter of the cerebral hemispheres and, to a lesser extent, in the brainstem and spinal cord white matter. Immunohistological examination and ultrastructural observations with an electron microscope suggest that these vacuolar lesions are remnants of degenerated oligodendrocytes. Electron microscopy also revealed myelin sheath destruction. Unexpectedly, aggregation of the four-repeat tau was observed in a spatial pattern reminiscent of progressive supranuclear palsy. The tau lesions included glial fibrillary tangles resembling tuft-shaped astrocytes and neurofibrillary tangles and pretangles. This is the first report to illustrate that a heterozygous missense mutation in ELOVL4 leads to neuronal loss accompanied by macrophages laden with PAS-positive material in the pontine base and oligodendroglial degeneration leading to widespread vacuoles in the white matter in SCA34.

Cholesterol-functionalized DNA/RNA heteroduplexes cross the blood-brain barrier and knock down genes in the rodent CNS.

Achieving regulation of endogenous gene expression in the central nervous system (CNS) with antisense oligonucleotides (ASOs) administered systemically would facilitate the development of ASO-based therapies for neurological diseases. We demonstrate that DNA/RNA heteroduplex oligonucleotides (HDOs) conjugated to cholesterol or α-tocopherol at the 5' end of the RNA strand reach the CNS after subcutaneous or intravenous administration in mice and rats. The HDOs distribute throughout the brain, spinal cord and peripheral tissues and suppress the expression of four target genes by up to 90% in the CNS, whereas single-stranded ASOs conjugated to cholesterol have limited activity. Gene knockdown was observed in major CNS cell types and was greatest in neurons and microglial cells. Side effects, such as thrombocytopenia and focal brain necrosis, were limited by using subcutaneous delivery or by dividing intravenous injections. By crossing the blood-brain barrier more effectively, cholesterol-conjugated HDOs may overcome the limited efficacy of ASOs targeting the CNS without requiring intrathecal administration.

Essential roles of plexin-B3+ oligodendrocyte precursor cells in the pathogenesis of Alzheimer's disease.

The role of oligodendrocyte lineage cells, the largest glial population in the adult central nervous system (CNS), in the pathogenesis of Alzheimer's disease (AD) remains elusive. Here, we developed a culture method for adult oligodendrocyte progenitor cells (aOPCs). Fibroblast growth factor 2 (FGF2) promotes survival and proliferation of NG2+ aOPCs in a serum-free defined medium; a subpopulation (~5%) of plexin-B3+ aOPCs was also found. FGF2 withdrawal decreased NG2+, but increased plexin-B3+ aOPCs and Aß1-42 secretion. Plexin-B3+ aOPCs were distributed throughout the adult rat brain, although less densely than NG2+ aOPCs. Spreading depolarization induced delayed cortical plexin-B3+ aOPC gliosis in the ipsilateral remote cortex. Furthermore, extracellular Aß1-42 accumulation was occasionally found around plexin-B3+ aOPCs near the lesions. In AD brains, virtually all cortical SPs were immunostained for plexin-B3, and plexin-B3 levels increased significantly in the Sarkosyl-soluble fractions. These findings suggest that plexin-B3+ aOPCs may play essential roles in AD pathogenesis, as natural Aß-secreting cells.

An autopsy case of corticobasal syndrome with pure diffuse Lewy Body Disease.

Corticobasal syndrome (CBS) is associated with diverse pathological substrates such as tau, prion protein, transactive response and, rarely, alpha synuclein. We report the case of a54-year-old man, who presented with asymmetric levodopa-poor-responsive parkinsonism, frontal lobe signs and behavioral changes. He was diagnosed with CBS, and postmortem analyses revealed Lewy body disease Braak stage VI without comorbid pathologies. Retrospectively, the clinical course of our patient and previous reports indicate that CBS plus mood changes and autonomic dysfunction, including reduced uptake of metaiodobenzylguanidine, are predictive factors of Lewy body pathology, even if the clinical picture is atypical.

Digital mapping of Lewy bodies and neurites in alpha-synuclein stained large cerebral hemispheric sections from three patients with dementia with Lewy bodies showing psychotic manifestations: A pilot study.

BACKGROUND: Dementia with Lewy bodies (DLB) is one of the major neurodegenerative diseases and a clinical diagnosis is made based on the fourth consensus report on DLB. However, clinicopathological features of DLB are variable among cases. METHODS: We analyzed three autopsy-proven cases of DLB (patients 1-3). Their clinical features were variable in spite of their pathological commonality. The entire hemispheric sections were stained for phosphorylated alpha-synuclein (aS), digitized, and each aS positive lesion was mapped using a virtual slide system. RESULTS: The three patients were clinically diagnosed as having DLB. However, patient 1 exhibited amnesia and misrecognition, while patient 3 exhibited abnormal behavior in addition to dementia. Therefore, both patients 1 and 3 did not fulfill the clinical criteria of DLB, in contrast to patient 2. In spite of the clinical heterogeneity, Lewy pathology was similar in patients 1, 2, and 3. Additionally, patient 1 exhibited less frequent Lewy neurites of the amygdala and entorhinal cortex, and less frequent neurofibrillary tangles and senile plaque as compared to patient 2. On the other hand, the Lewy pathology of patient 3 extended far beyond those of patients 1 and 2, wherein the superior to middle frontal cortices, insular cortex, and lentiform nucleus were severely affected by Lewy pathology. CONCLUSIONS: Clinical features could be variable among autopsy-proven cases of DLB. Furthermore, we show that the accent of Lewy pathology differs according to the variability of the clinical symptoms. This method will provide a comprehensive strategy to analyze wide-spread aS lesions scattered throughout the entire cerebral hemisphere and help determine the corresponding pathological lesions responsible for the clinical variability of neurodegenerative disorders, including DLB.

Acute Spinal Cord Infarction with Preferential Involvement of Ventral Gray Matter: An Autopsy Report.

Herein, we report abdominal aortic thrombosis as a rare cause of acute spinal cord infarction. A 78-year-old man with multiple vascular risk factors developed acute paraplegia with sensory and urinary disturbances and signs of ischemia in both lower limbs. The post-mortem study done 3 days after the onset of symptoms revealed a large coagulum in the abdominal aorta, distal to the renal arteries and extending to bilateral common iliac arteries; in addition, marked atherosclerosis was present in most large blood vessels. Premature incomplete necrotic foci were seen in the ventral gray matter of the spinal cord from T6 through S5; the surrounding white matter and dorsal gray matter were spared. Considering our autopsy case, spinal cord gray matter may be more vulnerable to ischemia than the white matter.