Investigating Neurovascular Function in Pre-Clinical Models of Alzheimer’s Disease & Atherosclerosis

Background: Neurovascular coupling (NVC) is essential to brain health and the breakdown of NVC is proposed to be a key pathological factor in the development of Alzheimer’s disease (AD), vascular dementia (VaD) and other cerebrovascular diseases. Importantly; as we age, the presence of two or more comorbidities is common and this often leads to clinical complications. Whilst preclinical models of human disease are numerous and have supported basic and translational neuroscience immensely over the past few decades, models of comorbidity are few and often neglected when it is important to study comorbidity to reflect clinical presentations in patients. This project will focus on examining neurovascular function in 3 different preclinical models of AD, atherosclerosis (ATH) and comorbid AD & ATH (MIX). Aims & Objectives: I) To investigate neurovascular function at an early-AD timepoint (6m) in the J20-hAPP model of AD (J20-AD); when amyloid-beta deposits begin to form, using a chronic surgery recovered animal protocol. Neurovascular function will be assessed by 2D- optical imaging spectroscopy (2D-OIS) to measure cortical haemodynamics, in addition to using multichannel microelectrodes to obtain neural multi-unit activity (MUA). II) To investigate neurovascular function in a novel experimental model of ATH using the rAAV8-mPCSK9- D377Y + Western Diet model (PCSK9-ATH). III) To create a mixed comorbid model of AD and ATH (J20-PCSK9-MIX) and to investigate neurovascular function in this novel model. IV) To assess neuropathology and neuroinflammation from brain tissue in the 3 disease models. Results: Firstly, at an early stage, J20-AD mice exhibit enhanced evoked-haemodynamic responses associated with neural hyperexcitability. They also display a unique time- dependent elevation of baseline blood volume under normobaric hyperoxia. Secondly, PCSK9-ATH display reduced evoked-responses and show signs of neurovascular dysfunction associated with increased IL1β & TNFα-neuroinflammation. Thirdly, J20-PCSK9-MIX comorbid mice have a trebling of Aβ plaques in the hippocampus, although, without any further worsening of neurovascular function in the cortex compared to J20-AD mice, although all 3 disease models show a trend towards the reduced washout of HbR, which indicates metabolic inefficiency and inadequate oxygen delivery to neurons. Finally, electrode insertion into the brain (causing mild brain injury) leads to cortical spreading depression (CSD) to occur in all mice, with the most severe CSD occurring in J20-AD and PCSK9-ATH mice, and this may be related to levels of IL1β neuroinflammation, though this needs to be confirmed. Conclusions: These results provide novel insights in all 3 disease models which have important translational implications by highlighting distinct therapeutic targets and strategies. The results also show the importance of neurovascular function in dementia and targeting impairments to neurovascular function early on may be key to slowing down the onset and progression of dementia

Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis

Background Paraspeckles are subnuclear bodies assembled on a long non-coding RNA (lncRNA) NEAT1. Their enhanced formation in spinal neurons of sporadic amyotrophic lateral sclerosis (ALS) patients has been reported but underlying mechanisms are unknown. The majority of ALS cases are characterized by TDP-43 proteinopathy. In current study we aimed to establish whether and how TDP-43 pathology may augment paraspeckle assembly. Methods Paraspeckle formation in human samples was analysed by RNA-FISH and laser capture microdissection followed by qRT-PCR. Mechanistic studies were performed in stable cell lines, mouse primary neurons and human embryonic stem cell-derived neurons. Loss and gain of function for TDP-43 and other microRNA pathway factors were modelled by siRNA-mediated knockdown and protein overexpression. Results We show that de novo paraspeckle assembly in spinal neurons and glial cells is a hallmark of both sporadic and familial ALS with TDP-43 pathology. Mechanistically, loss of TDP-43 but not its cytoplasmic accumulation or aggregation augments paraspeckle assembly in cultured cells. TDP-43 is a component of the microRNA machinery, and recently, paraspeckles have been shown to regulate pri-miRNA processing. Consistently, downregulation of core protein components of the miRNA pathway also promotes paraspeckle assembly. In addition, depletion of these proteins or TDP-43 results in accumulation of endogenous dsRNA and activation of type I interferon response which also stimulates paraspeckle formation. We demonstrate that human or mouse neurons in vitro lack paraspeckles, but a synthetic dsRNA is able to trigger their de novo formation. Finally, paraspeckles are protective in cells with compromised microRNA/dsRNA metabolism, and their assembly can be promoted by a small-molecule microRNA enhancer. Conclusions Our study establishes possible mechanisms behind paraspeckle hyper-assembly in ALS and suggests their utility as therapeutic targets in ALS and other diseases with abnormal metabolism of microRNA and dsRNA

Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis

BACKGROUND: Paraspeckles are subnuclear bodies assembled on a long non-coding RNA (lncRNA) NEAT1. Their enhanced formation in spinal neurons of sporadic amyotrophic lateral sclerosis (ALS) patients has been reported but underlying mechanisms are unknown. The majority of ALS cases are characterized by TDP-43 proteinopathy. In current study we aimed to establish whether and how TDP-43 pathology may augment paraspeckle assembly. METHODS: Paraspeckle formation in human samples was analysed by RNA-FISH and laser capture microdissection followed by qRT-PCR. Mechanistic studies were performed in stable cell lines, mouse primary neurons and human embryonic stem cell-derived neurons. Loss and gain of function for TDP-43 and other microRNA pathway factors were modelled by siRNA-mediated knockdown and protein overexpression. RESULTS: We show that de novo paraspeckle assembly in spinal neurons and glial cells is a hallmark of both sporadic and familial ALS with TDP-43 pathology. Mechanistically, loss of TDP-43 but not its cytoplasmic accumulation or aggregation augments paraspeckle assembly in cultured cells. TDP-43 is a component of the microRNA machinery, and recently, paraspeckles have been shown to regulate pri-miRNA processing. Consistently, downregulation of core protein components of the miRNA pathway also promotes paraspeckle assembly. In addition, depletion of these proteins or TDP-43 results in accumulation of endogenous dsRNA and activation of type I interferon response which also stimulates paraspeckle formation. We demonstrate that human or mouse neurons in vitro lack paraspeckles, but a synthetic dsRNA is able to trigger their de novo formation. Finally, paraspeckles are protective in cells with compromised microRNA/dsRNA metabolism, and their assembly can be promoted by a small-molecule microRNA enhancer. CONCLUSIONS: Our study establishes possible mechanisms behind paraspeckle hyper-assembly in ALS and suggests their utility as therapeutic targets in ALS and other diseases with abnormal metabolism of microRNA and dsRNA

Neurovascular dysfunction in vascular dementia, Alzheimer’s and atherosclerosis

Efficient blood supply to the brain is of paramount importance to its normal functioning and improper blood flow can result in potentially devastating neurological consequences. Cerebral blood flow in response to neural activity is intrinsically regulated by a complex interplay between various cell types within the brain in a relationship termed neurovascular coupling. The breakdown of neurovascular coupling is evident across a wide variety of both neurological and psychiatric disorders including Alzheimer’s disease. Atherosclerosis is a chronic syndrome affecting the integrity and function of major blood vessels including those that supply the brain, and it is therefore hypothesised that atherosclerosis impairs cerebral blood flow and neurovascular coupling leading to cerebrovascular dysfunction. This review will discuss the mechanisms of neurovascular coupling in health and disease and how atherosclerosis can potentially cause cerebrovascular dysfunction that may lead to cognitive decline as well as stroke. Understanding the mechanisms of neurovascular coupling in health and disease may enable us to develop potential therapies to prevent the breakdown of neurovascular coupling in the treatment of vascular brain diseases including vascular dementia, Alzheimer’s disease and stroke

Experimental study on fresh, mechanical properties and embodied carbon of concrete blended with sugarcane bagasse ash, metakaolin, and millet husk ash as ternary cementitious material

In recent years, there has been great concern about introducing new supplementary cementitious materials (SCM) in place of Portland cement (PC) in concrete. This study aims to investigate the behavior of sugarcane bagasse ash (SCBA), metakaolin (MK) and millet husk ash (MHA) as SCM with various proportions in concrete. The SCBA, MK and MHA are available in abundant quantities and considered as waste products. On the other hand, cement production emits a lot of toxic gases in the atmosphere which causes environmental pollution and greenhouse gases. Thus, SCBA, MK and MHA might be utilized as cementitious material in concrete for sustainable development. The effect of SCBA, MK and MHA as SCM on the fresh, mechanical properties and embodied carbon of concrete was evaluated experimentally. A total of 228 concrete specimens were prepared with targeted strength of 25MPa at 0.52 water-cement ratio and cured at 28 days. It is found that the compressive strength and split tensile strength were enhanced by 17% and 14.28% respectively at SCBA4MK4MHA4 (88% PC, 4% SCBA, 4% MK and 4% MHA) as ternary cementitious material (TCM) in concrete after 28 days. Moreover, the permeability and density of concrete are being reduced while utilizing of SCBA, MK and MHA separately as SCM and combined as TCM increases in concrete at 28 days respectively. Moreover, the workability of fresh concrete was decreased with the increase of the percentage of SCBA, MK and MHA separately as SCM and together as TCM in concrete. In addition to that, the use of SCBA, MK and MHA individually as SCM and combined as TCM in concrete can reduce the total carbon footprint while reducing the overall cost of concrete manufacturing

Vaccines to prevent pneumonia and improve child survival

For more than 30 years, vaccines have played an important part in pneumonia prevention. Recent advances have created opportunities for further improving child survival through prevention of childhood pneumonia by vaccination. Maximizing routine immunization with pertussis and measles vaccines, coupled with provision of a second opportunity for measles immunization, has rapidly reduced childhood deaths in low-income countries especially in sub-Saharan Africa

Neurovascular coupling preserved in a chronic mouse model of Alzheimer’s disease: Methodology is critical

Impaired neurovascular coupling has been suggested as an early pathogenic factor in Alzheimer’s disease (AD), which could serve as an early biomarker of cerebral pathology. We have established an anaesthetic regime to allow repeated measurements of neurovascular function over three months in the J20 mouse model of AD (J20-AD) and wild-type (WT) controls. Animals were 9-12 months old at the start of the experiment. Mice were chronically prepared with a cranial window through which optical imaging spectroscopy (OIS) was used to generate functional maps of the cerebral blood volume and saturation changes evoked by whisker stimulation and vascular reactivity challenges. Unexpectedly, the hemodynamic responses were largely preserved in the J20-AD group. This result failed to confirm previous investigations using the J20-AD model. However, a final acute electrophysiology and OIS experiment was performed to measure both neural and hemodynamic responses concurrently. In this experiment, previously reported deficits in neurovascular coupling in the J20-AD model were observed. This suggests that J20-AD mice may be more susceptible to the physiologically stressing conditions of an acute experimental procedure compared to WT animals. These results therefore highlight the importance of experimental procedure when determining the characteristics of animal models of human disease

Additional file 3: of Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis

Figure S3. Accumulation of dsRNA (a) and increased levels of p-eIF2α (b) in MCF7 cells depleted of TDP-43. Cells were analysed 48 h post-transfection. Scale bars, 100 μm and 10 μm for general plane and close-up panels respectively. (DOCX 663 kb

Additional file 1: of Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis

Figure S1. The effect of TDP-43 dysfunction on paraspeckles, speckles and paraspeckle proteins in MCF7 and SH-SY5Y cells. a and b TDP-43 siRNA-mediated knockdown upregulates NEAT1_2 (a) and enhances paraspeckle assembly (b) in SH-SY5Y cells. Cells were transfected with scrambled siRNA or Silencer® TDP-43 siRNA and analysed by qRT-PCR (n = 6). Mean number of paraspeckles per cell was also quantified using NEAT1_2 RNA FISH. **p < 0.01, ***p < 0.001 (Mann-Whitney U-test in a and Student’s t-test in b). c and d Downregulation of TDP-43 using an esiRNA (endoribonuclease-prepared MISSION® esiRNA, c) or shRNA (d) stimulates paraspeckle assembly. The efficiency of knockdown was analysed by TDP-43 immunocytochemistry, and paraspeckle assembly – by NEAT1_2 RNA-FISH. Representative images are shown. Scale bars, 100 μm for left panels and 10 μm for right panels. e Speckles visualised by MALAT1 RNA-FISH are not affected by TDP-43 knockdown. Representative images are shown. Scale bar, 10 μm. f Levels of core paraspeckle proteins NONO, SFPQ and FUS are not affected by TDP-43 knockdown in SH-SY5Y cells. Representative Western blots are shown. g Sequences and positions of gRNAs used for disrupting the NLS of the endogenous TDP-43 protein by CRISPR/Cas9-mediated editing. Two combinations of upstream and downstream gRNA sequences within TARDBP gene selected to disrupt the NLS are shown. The sequence encoding for the NLS is given in blue and PAM sites are boxed. h Transient transfection of two combinations of plasmids encoding upstream and downstream gRNAs for targeting the NLS of TDP-43 results in partial redistribution of endogenous TDP-43 but does not lead to enhanced paraspeckle formation. Cells were analysed 72 h post-transfection. Representative images are shown, asterisks indicate cells with cytoplasmic TDP-43 redistribution. Scale bar, 10 μm. In a-f, cells were analysed 48 h post-transfection. (DOCX 517 kb