The Therapeutic and Diagnostic Potential of Amyloid β Oligomers Selective Antibodies to Treat Alzheimer’s Disease

Improvements have been made in the diagnosis of Alzheimer’s disease (AD), manifesting mostly in the development of in vivo imaging methods that allow for the detection of pathological changes in AD by magnetic resonance imaging (MRI) and positron emission tomography (PET) scans. Many of these imaging methods, however, use agents that probe amyloid fibrils and plaques–species that do not correlate well with disease progression and are not present at the earliest stages of the disease. Amyloid β oligomers (AβOs), rather, are now widely accepted as the Aβ species most germane to AD onset and progression. Here we report evidence further supporting the role of AβOs as pathological instigators of AD and introduce promising anti-AβO diagnostic probes capable of distinguishing the 5xFAD mouse model from wild type mice by PET and MRI. In a developmental study, Aβ oligomers in 5xFAD mice were found to appear at 3 months of age, just prior to the onset of memory dysfunction, and spread as memory worsened. The increase of AβOs is prominent in the subiculum and correlates with concomitant development of reactive astrocytosis. The impact of these AβOs on memory is in harmony with findings that intraventricular injection of synthetic AβOs into wild type mice induced hippocampal dependent memory dysfunction within 24 h. Compelling support for the conclusion that endogenous AβOs cause memory loss was found in experiments showing that intranasal inoculation of AβO-selective antibodies into 5xFAD mice completely restored memory function, measured 30–40 days post-inoculation. These antibodies, which were modified to give MRI and PET imaging probes, were able to distinguish 5xFAD mice from wild type littermates. These results provide strong support for the role of AβOs in instigating memory loss and salient AD neuropathology, and they demonstrate that AβO selective antibodies have potential both for therapeutics and for diagnostics.

Cardiac-targeted PIASy Gene Silencing Mediates deSUMOylation of Caveolin-3 and Prevents Ischemia/reperfusion-induced Nav1.5 Down-regulation and Ventricular Arrhythmias

Background: Abnormal myocardial expression and function of Nav1.5 causes lethal ventricular arrhythmias during myocardial ischemia-reperfusion (I/R). PIASy mediated Caveolin-3 (Cav-3) SUMO modification affects Cav-3 binding to ligand Nav1.5. PIASy activity is increased after myocardial I/R, whether or not this may be attributable to plasma membrane Nav1.5 downregulation and ventricular arrhythmias remains unclear. Methods: Using recombinant adeno-associated virus subtype 9 (AAV9), rat cardiac PIASy was silenced by intraventricular injection of PIASy shRNA. Two weeks later, the hearts were subjected to I/R, and electrocardiography was performed to assess malignant arrhythmias. Tissues from peri-infarct areas of the left ventricle were collected for molecular biological measurement. Results: We found that PIASy was upregulated by I/R, with increased SUMO2/3 modification of Cav-3, reduced membrane Nav1.5 density, and increased ventricular arrhythmia frequency. These effects were significantly reversed by PIASy silencing. In addition, PIASy silencing enhanced Cav-3 binding to Nav1.5 and prevented I/R-induced Nav1.5 re-localization. Using in vitro models of HEK293T cells and isolated adult rat cardiomyocytes exposed to hypoxia/reoxygenation (H/R), this reserch further confirmed that PIASy promoted Cav-3 modification by SUMO2/3 and Nav1.5/Cav-3 dissociation after H/R. Mutation of the SUMO Consensus Sites Lysine in Cav-3 (K38R or K144R) alters the membrane expression levels of Nav1.5 and Cav-3 before and after H/R in HEK293T cells. Conclusions: I/R-induced cardiac PIASy activation contributes to Cav-3 SUMOylation by SUMO2/3 and dysregulated Nav1.5- related ventricular arrhythmias. Cardiac-targeted PIASy gene silencing mediates deSUMOylation of Cav-3 and prevents I/R-induced Nav1.5 down-regulation and ventricular arrhythmias in rats, identifying PIASy as a potential therapeutic target for relevant life-threatening arrhythmias in patients with ischemic heart diseases.

STEM-25. ADENO-ASSOCIATED VIRUS INTRAVENTRICULAR INJECTION ALLOWS THE RESTRICTED TRANSDUCTION OF GLIOBLASTOMA CELLS NESTED IN THE PERI- AND SUB-VENTRICULAR ZONE IN A PATIENT-DERIVED ORTHOTOPIC XENOGRAFT MODEL

INTRODUCTION: Glioblastoma (GBM) seems to arise from cells nested in the subventricular zone (SVZ), a neurogenic zone in the adult brain. After striatal engraftment of patient-derived GBM cells in mice, we showed that some tumor cells escape the tumor mass to colonize the SVZ. We aimed to determine if the SVZ-nested GBM cells could be highlighted by the intraventricular injection of a viral vector. MATERIALS AND METHODS: We determined the optimal Adeno-Associated Virus (AAV) serotype to transduce patient-derived GBM cells cultured in 3D (GB1). We studied the spreading of the virus 4 weeks after intraventricular injection in mice. We realized striatal engraftment with GB1 cells, previously transfected with a lentiviral construction in order to express the Red-Fluorescent-Protein (RFP) spontaneously, while they express Green-Fluorescent-Protein (eGFP) only in presence of Cre-recombinase. 10 weeks after engraftment, an AAV expressing Cre-Recombinase was injected in the lateral ventricle and mice were perfused at 14 weeks. RESULTS: AAV serotype DJ (AAVDJ) efficiently transduced GB1 cells. 4 weeks after intraventricular injection, the AAVDJ transduced cells in the SVZ and the medial part of the caudoputamen (mCP). The median of the longest distance between the right ventricle and the transduced cells in the mCP was 293.8µm [245 – 376.5]. In 5 mice, the median of the shortest distance between RFP-positive GBM cells and right ventricle was 580.1µm [535 – 785.1]. SVZ was not colonized and eGFP signal was not found. In the 4 other mice, SVZ was invaded and eGFP signal was detected. The median of the longest distance between the right ventricle and eGFP-positive tumor cells was 240.5µm [195.7 – 372.5]. The median of the eGFP volume was 9 499 404µm3. CONCLUSION: It is possible to transduce GBM cells nested in the peri- and sub-ventricular zone thanks to AAV intraventricular injection.

Prediction of persistent ventricular dilation by initial ventriculomegaly and clot volume in a porcine model

OBJECTIVE While intraventricular hemorrhage (IVH) is associated with posthemorrhagic ventricular dilation (PHVD), not all infants affected by high-grade IVH develop PHVD. The authors aimed to determine clot-associated predictors of PHVD in a porcine model by varying the amount and rate of direct intraventricular injection of whole autologous blood. METHODS Seven 1-week-old piglets underwent craniectomy and injection of autologous blood into the right lateral ventricle. They survived for a maximum of 28 days. MRI was performed prior to injection, immediately postoperatively, and every 7 days thereafter. T1-weighted, T2-weighted, and susceptibility-weighted imaging (SWI) sequences were used to segment ventricular and clot volumes. Spearman correlations were used to determine the relationship between blood and clot volumes and ventricular volumes over time. RESULTS The maximum ventricular volume was up to 12 times that of baseline. One animal developed acute hydrocephalus on day 4. All other animals survived until planned endpoints. The interaction between volume of blood injected and duration of injection was significantly associated with clot volume on the postoperative scan (p = 0.003) but not the amount of blood injected alone (p = 0.38). Initial postoperative and day 7 clot volumes, but not volume of blood injected, were correlated with maximum (p = 0.007 and 0.014) and terminal (p = 0.014 and 0.036) ventricular volumes. Initial postoperative ventricular volume was correlated with maximum and terminal ventricular volume (p = 0.007 and p = 0.014). CONCLUSIONS Initial postoperative, maximum, and terminal ventricular dilations were associated with the amount of clot formed, rather than the amount of blood injected. This supports the hypothesis that PHVD is determined by clot burden rather than the presence of blood products and allows further testing of early clot lysis to minimize PHVD risk.

Evaluation of the Effect of Resistance Exercise and Donepezil on Some Neurotrophin-Induced Changes in Gene Expression and Trk Receptors in the Hippocampus of Rats with Alzheimer’s Disease

Objectives: This study aimed to evaluate the impact of resistance exercise and donepezil on some neurotrophins gene expression and Trk receptors in the hippocampus of rats with Alzheimer’s disease (AD). Methods: In this study, 32 male adult Wistar rats (mean weight: 230 - 280 g) were assigned into two groups of AD and control. The control and AD groups received normal saline and streptozotocin (STZ) through intraventricular injection, respectively. Then, six subgroups were considered: (1) control rest (Con); (2) control exercise (Con-Exe); (3) Alzheimer’s rest (Alz); (4) Alzheimer’s exercise (Alz-Exe); (5) Alzheimer’s donepezil (Alz-Don); and (6) Alzheimer’s donepezil-exercise (Alz-Don-Exe). Donepezil was fed daily at a dose of 1.5 mg/kg to the treated groups. The three subgroups of exercising rats received exercises for eight weeks (three times a week). Each day, the resting groups were managed to decrease stress impacts. Twenty-four hours after the last session of exercise by the eighth week, deep anesthesia was applied, and the rats' heads were severed. Results: Considering an error rate below 5% (P < 0.05) and a confidence of more than 95%, a significant difference was observed in BDNF, NT3, NGF, TrkA, and TrkB values between exercising and donepezil-exercise rats compared to AD group. These values were considerably greater for donepezil-exercising Alzheimer’s group. Besides, the donepezil group was not significantly different from the Alzheimer’s group. Conclusions: Although the use of donepezil alone did not significantly increase the expression of the studied genes, the concomitant use of the drug and resistance training significantly increased the expression levels.

Amyloid β oligomer selective antibodies for Alzheimers therapeutics and diagnostics

Improvements have been made in the diagnosis of Alzheimer's disease (AD), manifesting mostly in the development of in vivo imaging methods that allow for the detection of pathological changes in AD by MRI and PET scans. Many of these imaging methods, however, use agents that probe amyloid fibrils and plaques- species that do not correlate well with disease progression and are not present at the earliest stages of the disease. Amyloid β oligomers (AβOs), rather, are now widely accepted as the Aβ species most germane to AD onset and progression. Here we report evidence further supporting the role of AβOs as pathological instigators of AD and introduce a promising anti-AβO diagnostic probe capable of distinguishing the 5xFAD mouse model from wild type mice by PET and MRI. In a developmental study, Aβ oligomers in 5xFAD mice were found to appear at 3 months of age, just prior to the onset of memory dysfunction, and spread as memory worsened. The increase is prominent in the subiculum and correlates with concomitant development of reactive astrocytosis. The impact of these AβOs on memory is in harmony with findings that intraventricular injection of synthetic AβOs into wild type mice induced hippocampal dependent memory dysfunction within 24 hours. Compelling support for the conclusion that endogenous AβOs cause memory loss was found in experiments showing that intranasal inoculation of AβO-selective antibodies into 5xFAD mice completely restored memory function, measured 30 days post-inoculation. These antibodies, which were modified to give MRI and PET imaging probes, were able to distinguish 5xFAD mice from wild type littermates. These results provide strong support for the role of AβOs in instigating memory loss and salient AD neuropathology, and they demonstrate that AβO selective antibodies have potential both for therapeutics and for diagnostics.

Development and Validation of an HPLC Method for Analysis of Topotecan in Human Cerebrospinal Fluid and Its Application in Elimination Evaluation of Topotecan after Intraventricular Injection

Intrathecal administration of anticancer drugs is an effective dosage strategy, but the elimination of intraventricular drugs is not uniform in all patients. For safety, a system to evaluate local pharmacokinetics in the ventricles after administration is desired. In this study, we developed a simple and reproducible method to measure topotecan concentration in the cerebrospinal fluid (CSF) and confirmed its clinical applicability. High-performance liquid chromatography (HPLC) analysis was performed using a C18 column to measure the total topotecan concentration in the CSF. Clinical CSF samples were obtained from a 1-year old child with poor CSF absorption and stagnation. The patient received topotecan via an intraventricular subcutaneous reservoir. The HPLC method complied with the validation criteria. The lower limit of quantitation of this method was 0.04 µM. Using the developed method, we could determine the difference in topotecan CSF concentrations at 24 and 48 h after administration. The patient’s topotecan elimination rate was extremely low, and signs of adverse effects were observed at high CSF concentration of topotecan. The developed method could detect the delay in topotecan elimination after intrathecal injection. The findings of this study are valuable for the development of personalized treatments for the intrathecal administration of anticancer drugs.

Hypercholesterolemia and 27-Hydroxycholesterol Increase S100A8 and RAGE Expression in the Brain: a Link Between Cholesterol, Alarmins, and Neurodegeneration

Alterations in cholesterol metabolism in the brain have a major role in the physiology of Alzheimer’s disease (AD). Oxysterols are cholesterol metabolites with multiple implications in memory functions and in neurodegeneration. Previous studies have shown detrimental effects of cholesterol metabolites in neurons, but its effect in glial cells is unknown. We used a high-fat/high-cholesterol diet in mice to study the effects of hypercholesterolemia over the alarmin S100A8 cascade in the hippocampus. Using CYP27Tg, a transgenic mouse model, we show that the hypercholesterolemia influence on the brain is mediated by the excess of 27-hydroxycholesterol (27-OH), a cholesterol metabolite. We also employed an acute model of 27-OH intraventricular injection in the brain to study RAGE and S100A8 response. We used primary cultures of neurons and astrocytes to study the effect of high levels of 27-OH over the S100A8 alarmin cascade. We report that a high-fat/high-cholesterol diet leads to an increase in S100A8 production in the brain. In CYP27Tg, we report an increase of S100A8 and its receptor RAGE in the hippocampus under elevated 27-OH in the brain. Using siRNA, we found that 27-OH upregulation of RAGE in astrocytes and neurons is mediated by the nuclear receptor RXRγ. Silencing RXRγ in neurons prevented 27-OH-mediated upregulation of RAGE. These results show that S100A8 alarmin and RAGE respond to high levels of 27-OH in the brain in both neurons and astrocytes through RXRγ. Our study supports the notion that 27-OH mediates detrimental effects of hypercholesterolemia to the brain via alarmin signaling.

Gingival and intraventricular haemorrhages are severe newborn diseases causing damage to white matter and neurological dysfunction in surviving newborns who can benefit from gene therapy

Gingival matrix haemorrhage (GMH) and intraventricular haemorrhage (IVH) are major neonatal disorders causing white matter damage (WMI) and neurological impairments in surviving neonates. Preclinical and clinical IVH research has advanced our understanding of the mechanisms driving IVH-induced WMI, including negative effects on oligodendrocyte progenitor cells (OPCs) and blood-generated axons, especially iron and thrombin.Right now, rehabilitation is the sole therapy option. As a result, new treatments must be developed faster to boost these neonates' outcomes. The most promising therapies in IVH124,147,151 infants are blood clot clearance (DRIFT or endoscopic lavage), early intervention (ELVIS), and stem cell transplantation.However, several barriers must be overcome before the findings of this research can be utilized in clinical practice, as both surgical intervention and intraventricular injection have inherent hazards and adverse effects. Brain damage is projected to be decreased by thorough neuro-endoscopic ventricular lavage surgery, and the optimal distribution strategy for stem cells is projected to enhance their therapeutic advantages. Therapy must begin before scarring and permanent damage develop in neonates with IVH, so determining the appropriate treatment window is critical for success.DRIFT clinical research was carried out in newborns with IVH at a median age of 20 days, and some improvements were found, but not as many as expected147.The median age of intervention in ELVIS was 9 days, leading to the lowest ventriculoperitoneal shunt rate observed in neonates with IVH to date and superior results at 2 years111. Nevertheless, prudence is suggested since early surgery may worsen IVH and aggravate damage.Several mechanism-targeted therapies were explored in preclinical animals. Oral or intramuscular thyroxine or celecoxib appear to be the most promising therapy in animal studies, as these drugs were previously granted to humans by the FDA, are straightforward to give and have low adverse-effect profiles. However, iron chelators or anti-thrombin treatment may be poorly tolerated and associated with significant adverse effects.Because the population of people with IVH is so different, a tailored approach to therapy selection may be needed. Blood clot removal, for example, is suitable for babies with grade III IVH but not isolated PVHI. Similarly, regenerative treatments and agents that reduce inflammation and oxidative stress in the brain (such as celecoxib, antioxidants, thyroxine, and stem cells) are likely to be most effective in infants with moderate to severe IVH, while the risk of side effects may limit their use in infants with IVH grade. Other frequent concerns in premature neonates include severe respiratory distress syndrome, low blood pressure, infection, patent ductus arteriosus, and low platelet levels, all of which may delay treatment. Despite these hurdles, feasible therapeutic solutions for neonates with IVH do not seem too far away.

Intraoperative ventricular volume restoration by intraventricular Ringer solution injection in a normal-pressure hydrocephalus patient with traumatic bilateral acute subdural hematoma and ventricular system collapse caused by cerebrospinal fluid shunt overdrainage: illustrative case

BACKGROUND The authors report a case of a patient with normal-pressure hydrocephalus treated with a ventriculoperitoneal shunt who developed a traumatic hemispheric bilateral acute subdural hematoma caused by quick cerebrospinal fluid (CSF) overdrainage. The authors present active ventricular CSF volume restoration as a novel treatment option. CSF overdrainage in patients with shunts may facilitate acute subdural hematoma formation even in cases of minor head trauma. Therapeutic options include CSF shunt function restriction or ligation, hematoma evacuation, or a combination of both. OBSERVATIONS In this case, the authors performed emergency surgery with hematoma evacuation through a bilateral craniotomy and actively restored the volume of the ventricular system with a slow intraventricular injection of 37°C warmed Ringer solution through a shunt burr hole reservoir. LESSONS In addition to hematoma evacuation and restriction of shunt function, the intraoperative restoration of ventricular volume could be a treatment option to prevent postoperative rebleeding or a space-occupying air collection in the subdural space. The risk of possible complications, such as ventricular or parenchymal bleeding, shunt dysfunction, or infections, requires further investigation. Restoration of the ventricular CSF volume by intraventricular injection of Ringer solution was in this case an efficient treatment method to prevent subdural rebleeding and a space-occupying air collection after subdural hematoma evacuation.