Alan King Lun Liu

Nucleus basalis of Meynert revisited: anatomy, history and differential involvement in Alzheimer’s and Parkinson’s disease

It has been well established that neuronal loss within the cholinergic nucleus basalis of Meynert (nbM) correlates with cognitive decline in dementing disorders such as Alzheimer’s disease (AD). Friedrich Lewy first observed his eponymous inclusion bodies in the nbM of postmortem brain tissue from patients with Parkinson’s disease (PD) and cell loss in this area can be at least as extensive as that seen in AD. There has been confusion with regard to the terminology and exact localisation of the nbM within the human basal forebrain for decades due to the diffuse and broad structure of this “nucleus”. Also, while topographical projections from the nbM have been mapped out in subhuman primates, no direct clinicopathological correlations between subregional nbM and cortical pathology and specific cognitive profile decline have been performed in human tissue. Here, we review the evolution of the term nbM and the importance of standardised nbM sampling for neuropathological studies. Extensive review of the literature suggests that there is a caudorostral pattern of neuronal loss within the nbM in AD brains. However, the findings in PD are less clear due to the limited number of studies performed. Given the differing neuropsychiatric and cognitive deficits in Lewy body-associated dementias (PD dementia and dementia with Lewy bodies) as compared to AD, we hypothesise that a different pattern of neuronal loss will be found in the nbM of Lewy body disease brains. Understanding the functional significance of the subregions of the nbM could prove important in elucidating the pathogenesis of dementia in PD.

Bringing CLARITY to the human brain: visualization of Lewy pathology in three dimensions.

CLARITY is a novel technique which enables three‐dimensional visualization of immunostained tissue for the study of circuitry and spatial interactions between cells and molecules in the brain. In this study, we aimed to compare methodological differences in the application of CLARITY between rodent and large human post mortem brain samples. In addition, we aimed to investigate if this technique could be used to visualize Lewy pathology in a post mortem Parkinsons brain.

ARTAG in the basal forebrain: Widening the constellation of astrocytic tau pathology

Tauopathies are disorders characterised by the abnormal accumulation of hyperphosphorylated tau protein within neurons and glial cells. Alzheimer’s disease (AD), the most common form of tauopathy, features a stereotypical, staged progression of neurofibrillary tangle (NFT) and neuritic tau pathology through the brain [1]. However, age-related NFT and neuritic tau pathologies have also commonly been identified in cognitively normal and ‘tangle-only dementia’ cases in the absence of amyloid-β peptide (Aβ) pathology. This has now been given the term ‘primary age-related tauopathy’ (PART) to distinguish it from AD [3]. Furthermore, astroglial tau aggregations have been increasingly recognised to be present within the aging brain independently of any co-existing neuropathological disorders or cognitive impairment. This unique astroglial tau pathology has been termed aging-related tau astrogliopathy (ARTAG) [6]. ARTAG exists in two distinct morphological forms as thorn-shaped astrocytes (TSA) and granular/fuzzy astrocytes (GFA). In addition, these tau immunoreactive astrocytes show a unique distribution within the brain, with TSA and/or GFA commonly found in subpial, subependymal and perivascular locations, as well as in clusters within the white and grey matter.

Free-of-acrylamide SDS-based Tissue Clearing (FASTClear): A novel protocol of tissue clearing for three-dimensional visualisation of human brain tissues

In recent years, advances in laser microscopy and endogenous fluorescent tagging techniques has led to the development of many tissue clearing strategies, which render tissues optically transparent, allowing large blocks of un-sectioned tissue to be visualised in three-dimensions (3D). CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel) is one of the tissue clearing techniques which works by fixation/hybridisation of brain tissue using hydrogel crosslinks and subsequent detergent-based delipidation to turn the tissue transparent [1]. Since CLARITY enables molecular probing using immunofluorescence, this technique was deemed suitable for post-mortem human brain tissues to demonstrate the potential in visualising pathologies in Alzheimers [2], Parkinsons [3] and neurodevelopmental disorders [1,4] in 3D. Early attempts using CLARITY were performed on cortical tissue from the brains of children [1,4], which is considered to be less myelinated than adults, or on thinly sectioned tissues of up to 500 μm in thickness [1,2]. This article is protected by copyright. All rights reserved.

Differential expression of galanin in the cholinergic basal forebrain of patients with Lewy body disorders

IntroductionDepletion of cholinergic neurons within the nucleus basalis of Meynert (nbM) is thought to contribute to the development of cognitive impairments in both Alzheimer’s disease (AD) and Lewy body disorders (LBD). It has been reported that, in late stage AD, a network of fibres that contain the neuropeptide galanin displays significant hypertrophy and ‘hyperinnervates’ the surviving cholinergic neurons. Galanin is considered as a highly inducible neuroprotective factor and in AD this is assumed to be part of a protective tissue response. The aim of this study was to determine if a similar galanin upregulation is present in the nbM in post-mortem tissue from patients with LBD. Gallatin immunohistochemistry was carried out on anterior nbM sections from 76 LBD cases (27 PD, 15 PD with mild cognitive impairment (MCI), 34 PD with dementia (PDD) and 4 aged-matched controls. Galaninergic innervation of cholinergic neurons was assessed on a semi-quantitative scale.ResultsThe LBD group had significantly higher galaninergic innervation scores (p = 0.016) compared to controls. However, this difference was due to increased innervation density only in a subgroup of LBD cases and this correlated positively with choline acetyltransferase–immunopositive neuron density.ConclusionGalanin upregulation within the basal forebrain cholinergic system in LBD, similar to that seen in AD, may represent an intrinsic adaptive response to neurodegeneration that is consistent with its proposed roles in neurogenesis and neuroprotection.

Next generation histology methods for three-dimensional imaging of fresh and archival human brain tissues

Modern clearing techniques for the three-dimensional (3D) visualisation of neural tissue microstructure have been very effective when used on rodent brain but very few studies have utilised them on human brain material, mainly due to the inherent difficulties in processing post-mortem tissue. Here we develop a tissue clearing solution, OPTIClear, optimised for fresh and archival human brain tissue, including formalin-fixed paraffin-embedded material. In light of practical challenges with immunostaining in tissue clearing, we adapt the use of cresyl violet for visualisation of neurons in cleared tissue, with the potential for 3D quantification in regions of interest. Furthermore, we use lipophilic tracers for tracing of neuronal processes in post-mortem tissue, enabling the study of the morphology of human dendritic spines in 3D. The development of these different strategies for human tissue clearing has wide applicability and, we hope, will provide a baseline for further technique development.Current available tissue clearing techniques are mostly used for rodent tissues. Here, the authors develop OPTIClear solution for fresh and archival human brain tissue clearing and establish associated protocols for three-dimensional histological investigations.

Neuropathological Assessment as an Endpoint in Clinical Trial Design

Different neurodegenerative conditions can have complex, overlapping clinical presentations that make accurate diagnosis during life very challenging. For this reason, confirmation of the clinical diagnosis still requires postmortem verification. This is particularly relevant for clinical trials of novel therapeutics where it is important to ascertain what disease and/or pathology modifying effects the therapeutics have had. Furthermore, it is important to confirm that patients in the trial actually had the correct clinical diagnosis as this will have a major bearing on the interpretation of trial results. Here we present a simple protocol for pathological assessment of neurodegenerative changes.

Review: Revisiting the human cholinergic nucleus of the diagonal band of Broca

Although the nucleus of the vertical limb of the diagonal band of Broca (nvlDBB) is the second largest cholinergic nucleus in the basal forebrain, after the nucleus basalis of Meynert, it has not generally been a focus for studies of neurodegenerative disorders. However, the nvlDBB has an important projection to the hippocampus and discrete lesions of the rostral basal forebrain have been shown to disrupt retrieval memory function, a major deficit seen in patients with Lewy body disorders. One reason for its neglect is that the anatomical boundaries of the nvlDBB are ill defined and this area of the brain is not part of routine diagnostic sampling protocols. We have reviewed the history and anatomy of the nvlDBB and now propose guidelines for distinguishing nvlDBB from other neighbouring cholinergic cell groups for standardizing future clinicopathological work. Thorough review of the literature regarding neurodegenerative conditions reveals inconsistent results in terms of cholinergic neuronal loss within the nvlDBB. This is likely to be due to the use of variable neuronal inclusion criteria and omission of cholinergic immunohistochemical markers. Extrapolating from those studies showing a significant nvlDBB neuronal loss in Lewy body dementia, we propose an anatomical and functional connection between the cholinergic component of the nvlDBB (Ch2) and the CA2 subfield in the hippocampus which may be especially vulnerable in Lewy body disorders.

Author Correction: Next generation histology methods for three-dimensional imaging of fresh and archival human brain tissues

In the original version of this Article, the concentration of boric acid buffer for the SDS clearing solution was given incorrectly as ‘1 M sodium borate’ and should have read ‘0.2 M boric acid’. Also, the composition of PBST incorrectly read ‘1% Triton X-100 (vol/vol) and 0.1% sodium azide (wt/vol)’ and should have read ‘0.1% Triton X-100 (vol/vol) and 0.01% sodium azide (wt/vol)’. Further, the pH of the OPTIClear solution was not stated, and should have read ‘with a pH between 7 to 8 adjusted with hydrochloric acid’. These errors have been corrected in both the PDF and HTML versions of the Article.