Daniel Blackburn

Astrocyte Function and Role in Motor Neuron Disease: A Future Therapeutic Target?

Astrocytes are the most numerous cell type within the central nervous system (CNS) and perform a variety of tasks, from axon guidance and synaptic support, to the control of the blood brain barrier and blood flow. To perform these roles, there is a great variety of astrocytes. In this review, we summarize the function of astrocytes, in particular, their role in maintaining homeostasis at the synapse, regulating neuronal signaling, protecting neurons from oxidative damage, and determining the fate of endogenous neural precursors. The review also highlights recent developments indicating the role of astrocytes in motor neuron disease (MND), emphasizing their potential as therapeutic targets and agents in cell replacement therapy. The Cu‐Zn superoxide dismutase (SOD1) gene that has been implicated in 20% of cases of familial MND must be expressed in the glial cells as well as motor neurons to produce the disease state in murine models of disease. Selectively reducing mutant SOD1 (mSOD1) in astrocytes does not affect disease onset but slows disease progression, whereas reducing mSOD1 in motor neurons delays disease onset and slows early disease but has less effect on life span. This suggests that glial cells represent potential therapeutic targets in MND. However, the lack of specific markers for astrocytes, their precursors, and sub‐types means that our knowledge of astrocyte development/differentiation and response to injury lags far behind our understanding of function. Only by filling this knowledge gap can astrocytes be effectively targeted or replaced to successfully treat chronic CNS disorders such as MND.

Mutant SOD1 G93A microglia have an inflammatory phenotype and elevated production of MCP-1.

The inflammatory response in amyotrophic lateral sclerosis (ALS) is well documented but the underlying cellular mechanisms have not been fully elucidated. We report that microglia isolated from the mutant human superoxide dismutase 1 (SOD1) G93A transgenic mouse model of ALS have an increased response to the inflammatory stimulus, lipopolysaccharide. Cell surface area and F4/80 surface marker, both indicators of cell activation, are increased relative to transgenic wild-type human SOD1 microglia. Monocyte chemoattractant protein-1, known to be increased in ALS, is produced at three-fold higher levels by SOD1 G93A than by wild-type human SOD1 microglia, under activating conditions. This novel finding implicates ALS microglia as a source of the increased monocyte chemoattractant protein-1 levels detected in ALS patients and in the ALS mouse model.

Memory difficulties are not always a sign of incipient dementia: a review of the possible causes of loss of memory efficiency

INTRODUCTION OR BACKGROUND Memory problems are a very common reason for presenting to primary care. There is a need for better treatments for dementia. Increased government and media interest may result in greater number seeking help for memory problems, which may not reduce the dementia gap but rather increase numbers seen who do not have dementia. This review highlights the issues around the diagnostic criteria and terminology used for people with memory complaints. SOURCES OF DATA A comprehensive literature search using PubMed using keywords for articles on subjective memory decline (SMD)/impairment/complaints, subjective cognitive decline (SCD), mild cognitive impairment (MCI) and functional memory disorder (FMD). AREAS OF AGREEMENT There is a need for early accurate detection of dementia syndromes so that trials of new treatments can begin earlier on the disease process. AREAS OF CONTROVERSY Diagnostic criteria and terminology used for disorders of memory including SCD, MCI and FMD. GROWING POINTS This article reviews SCD and whether this can be used to predict Alzheimers disease. The review also discusses the terminology used for non-progressive memory problems and the long-term outcomes for this patient group. AREAS TIMELY FOR DEVELOPING RESEARCH The accurate distinction of premorbid dementia syndromes from benign non-progressive memory problems. Studies of treatment options for people with benign non-progressive memory problems and longer-term follow-up to determine which patients develop chronic problems.

Conversational assessment in memory clinic encounters: interactional profiling for differentiating dementia from functional memory disorders

Objectives: In the UK dementia is under-diagnosed, there is limited access to specialist memory clinics, and many of the patients referred to such clinics are ultimately found to have functional (non-progressive) memory disorders (FMD), rather than a neurodegenerative disorder. Government initiatives on ‘timely diagnosis’ aim to improve the rate and quality of diagnosis for those with dementia. This study seeks to improve the screening and diagnostic process by analysing communication between clinicians and patients during initial specialist clinic visits. Establishing differential conversational profiles could help the timely differential diagnosis of memory complaints. Method: This study is based on video- and audio recordings of 25 initial consultations between neurologists and patients referred to a UK memory clinic. Conversation analysis was used to explore recurrent communicative practices associated with each diagnostic group. Results: Two discrete conversational profiles began to emerge, to help differentiate between patients with dementia and functional memory complaints, based on (1) whether the patient is able to answer questions about personal information; (2) whether they can display working memory in interaction; (3) whether they are able to respond to compound questions; (4) the time taken to respond to questions; and (5) the level of detail they offer when providing an account of their memory failure experiences. Conclusion: The distinctive conversational profiles observed in patients with functional memory complaints on the one hand and neurodegenerative memory conditions on the other suggest that conversational profiling can support the differential diagnosis of functional and neurodegenerative memory disorders.

A comparison of in vitro properties of resting SOD1 transgenic microglia reveals evidence of reduced neuroprotective function

BackgroundOverexpression of mutant copper/zinc superoxide dismutase (SOD1) in rodents has provided useful models for studying the pathogenesis of amyotrophic lateral sclerosis (ALS). Microglia have been shown to contribute to ALS disease progression in these models, although the mechanism of this contribution remains to be elucidated. Here, we present the first evidence of the effects of overexpression of mutant (TG G93A) and wild type (TG WT) human SOD1 transgenes on a set of functional properties of microglia relevant to ALS progression, including expression of integrin β-1, spreading and migration, phagocytosis of apoptotic neuronal cell debris, and intracellular calcium changes in response to an inflammatory stimulus.ResultsTG SOD1 G93A but not TG SOD1 WT microglia had lower expression levels of the cell adhesion molecule subunit integrin β-1 than their NTG control cells [NTG (G93A) and NTG (WT), respectively, 92.8 ± 2.8% on TG G93A, 92.0 ± 6.6% on TG WT, 100.0 ± 1.6% on NTG (G93A), and 100.0 ± 2.7% on NTG (WT) cells], resulting in decreased spreading ability, with no effect on ability to migrate. Both TG G93A and TG WT microglia had reduced capacity to phagocytose apoptotic neuronal cell debris (13.0 ± 1.3% for TG G93A, 16.5 ± 1.9% for TG WT, 28.6 ± 1.8% for NTG (G93A), and 26.9 ± 2.8% for NTG (WT) cells). Extracellular stimulation of microglia with ATP resulted in smaller increase in intracellular free calcium in TG G93A and TG WT microglia relative to NTG controls (0.28 ± 0.02 μM for TG G93A, 0.24 ± 0.03 μM for TG WT, 0.39 ± 0.03 μM for NTG (G93A), and 0.37 ± 0.05 μM for NTG (WT) microglia).ConclusionsThese findings indicate that, under resting conditions, microglia from mutant SOD1 transgenic mice have a reduced capacity to elicit physiological responses following tissue disturbances and that higher levels of stimulatory signals, and/or prolonged stimulation may be necessary to initiate these responses. Overall, resting mutant SOD1-overexpressing microglia may have reduced capacity to function as sensors of disturbed tissue/cellular homeostasis in the CNS and thus have reduced neuroprotective function.

Lysosomal and phagocytic activity is increased in astrocytes during disease progression in the SOD1 G93A mouse model of amyotrophic lateral sclerosis

Astrocytes are key players in the progression of amyotrophic lateral sclerosis (ALS). Previously, gene expression profiling of astrocytes from the pre-symptomatic stage of the SOD1G93A model of ALS has revealed reduced lactate metabolism and altered trophic support. Here, we have performed microarray analysis of symptomatic and late-stage disease astrocytes isolated by laser capture microdissection (LCM) from the lumbar spinal cord of the SOD1G93A mouse to complete the picture of astrocyte behavior throughout the disease course. Astrocytes at symptomatic and late-stage disease show a distinct up-regulation of transcripts defining a reactive phenotype, such as those involved in the lysosome and phagocytic pathways. Functional analysis of hexosaminidase B enzyme activity in the spinal cord and of astrocyte phagocytic ability has demonstrated a significant increase in lysosomal enzyme activity and phagocytic activity in SOD1G93A vs. littermate controls, validating the findings of the microarray study. In addition to the increased reactivity seen at both stages, astrocytes from late-stage disease showed decreased expression of many transcripts involved in cholesterol homeostasis. Staining for the master regulator of cholesterol synthesis, SREBP2, has revealed an increased localization to the cytoplasm of astrocytes and motor neurons in late-stage SOD1G93A spinal cord, indicating that down-regulation of transcripts may be due to an excess of cholesterol in the CNS during late-stage disease possibly due to phagocytosis of neuronal debris. Our data reveal that SOD1G93A astrocytes are characterized more by a loss of supportive function than a toxic phenotype during ALS disease progression and future studies should focus upon restorative therapies.

Intensive versus guideline blood pressure and lipid lowering in patients with previous stroke: main results from the pilot ‘Prevention Of Decline in Cognition After Stroke Trial’ (PODCAST) randomised controlled trial

Background Stroke is associated with the development of cognitive impairment and dementia. We assessed the effect of intensive blood pressure (BP) and/or lipid lowering on cognitive outcomes in patients with recent stroke in a pilot trial. Methods In a multicentre, partial-factorial trial, patients with recent stroke, absence of dementia, and systolic BP (SBP) 125–170 mmHg were assigned randomly to at least 6 months of intensive (target SBP <125 mmHg) or guideline (target SBP <140 mmHg) BP lowering. The subset of patients with ischaemic stroke and total cholesterol 3.0–8.0 mmol/l were also assigned randomly to intensive (target LDL-cholesterol <1.3 mmol/l) or guideline (target LDL-c <3.0 mmol/l) lipid lowering. The primary outcome was the Addenbrooke’s Cognitive Examination-Revised (ACE-R). Results We enrolled 83 patients, mean age 74.0 (6.8) years, and median 4.5 months after stroke. The median follow-up was 24 months (range 1–48). Mean BP was significantly reduced with intensive compared to guideline treatment (difference –10·6/–5·5 mmHg; p<0·01), as was total/LDL-cholesterol with intensive lipid lowering compared to guideline (difference –0·54/–0·44 mmol/l; p<0·01). The ACE-R score during treatment did not differ for either treatment comparison; mean difference for BP lowering -3.6 (95% CI -9.7 to 2.4), and lipid lowering 4.4 (95% CI -2.1 to 10.9). However, intensive lipid lowering therapy was significantly associated with improved scores for ACE-R at 6 months, trail making A, modified Rankin Scale and Euro-Qol Visual Analogue Scale. There was no difference in rates of dementia or serious adverse events for either comparison. Conclusion In patients with recent stroke and normal cognition, intensive BP and lipid lowering were feasible and safe, but did not alter cognition over two years. The association between intensive lipid lowering and improved scores for some secondary outcomes suggests further trials are warranted. Trial Registration ISRCTN ISRCTN85562386

Autologous hematopoietic stem cell transplantation following pulsed cyclophosphamide in a severely disabled patient with malignant multiple sclerosis

Multiple sclerosis (MS) may run a rapidly progressive monophasic course (the Marburg, or malignant form), that carries a poor prognosis [1]. Autologous hematopoietic stem cell transplant (AHSCT), initially utilised in chronic MS [2], is now occasionally administered in acute aggressive relapsing–remitting MS (RRMS) [3]. We describe a case of malignant-type MS treated with pulsed cyclophosphamide (CY) and non-myeloablative AHSCT. A 21-year-old woman, presented with sensorimotor disturbance to all four limbs in April 2011. Neuro-imaging demonstrated demyelination with enhancement (Fig. 1a), cerebrospinal fluid (CSF) demonstrated 47 lymphocytes and was positive for oligoclonal bands. Serum was negative for aquporin4 (Aq4) antibodies. Three days of 1 g IV methylprednisolone (IVMP) failed to slow progression. A second course of IVMP 5 weeks later was unsuccessful, as were seven sessions of plasma exchange undertaken over a 2-week period and the patient became tetraplegic. A 5-day course of alemtuzumab with pulsed IVMP was completed (week 9) but deterioration continued with decreased visual acuity (right eye: counting fingers, left eye: 6/36), vertigo and respiratory compromise requiring ventilatory support. MRI demonstrated new lesions (Fig. 1b). Repeat CSF revealed 10 lymphocytes and Aq4 was negative in serum and CSF. Alemtuzumab can take up to 3 months to take effect, however, in July 2011 (13 weeks after presentation) treatment was intensified due to continued decline and stem cells mobilised with CY 2 g/m plus G-CSF (10 lg/kg/day). Stem cell harvest was undertaken 2 weeks later. A second dose of CY (2 g/m) 4 weeks poststem cell harvest was followed by improvement—return of sensorimotor function to the left arm, cessation of vertigo and weaning from ventilatory support. Two months after first treatment with CY the patient underwent AHSCT using CY 200 mg/kg ? rabbit antithymocyte globulin (ATG, total dose 6 mg/kg). At this point she had an Expanded Disability Status Score (EDSS) of 8.5. Routine toxicity, including neutropenic sepsis, was managed and engraftment prompt. Four months from AHSCT, she could walk a few steps using parallel bars and had visual acuities of 6/24 (right) and 6/9 (left); EDSS 7.5. Repeat MRI demonstrated a stable lesion load (Fig. 1c). One year from the initiation of treatment with CY (8 months from AHSCT) she was able to walk 10 M with bilateral support, EDSS 6.5. Further imaging demonstrated no change. EBMT guidance [4] advises that patients with an EDSS score higher than 6.5 be excluded from AHSCT, except in instances of ‘malignant’ MS. Our patient had a rapid-onset demyelinating illness that we have termed malignant MS. We cannot exclude neuromyelitis optica (NMO), although Aq4 antibodies were consistently negative, including in CSF. In such cases, the distinction between MS, NMO and J. J. P. Alix (&) D. J. Blackburn B. Sharrack Sheffield Institute for Translational Neuroscience, University of Sheffield, 385A Glossop Road, Sheffield S10 2HQ, UK e-mail: j.alix@sheffield.ac.uk

Toward the Automation of Diagnostic Conversation Analysis in Patients with Memory Complaints

BACKGROUND The early diagnosis of dementia is of great clinical and social importance. A recent study using the qualitative methodology of conversation analysis (CA) demonstrated that language and communication problems are evident during interactions between patients and neurologists, and that interactional observations can be used to differentiate between cognitive difficulties due to neurodegenerative disorders (ND) or functional memory disorders (FMD). OBJECTIVE This study explores whether the differential diagnostic analysis of doctor-patient interactions in a memory clinic can be automated. METHODS Verbatim transcripts of conversations between neurologists and patients initially presenting with memory problems to a specialist clinic were produced manually (15 with FMD, and 15 with ND). A range of automatically detectable features focusing on acoustic, lexical, semantic, and visual information contained in the transcripts were defined aiming to replicate the diagnostic qualitative observations. The features were used to train a set of five machine learning classifiers to distinguish between ND and FMD. RESULTS The mean rate of correct classification between ND and FMD was 93% ranging from 97% by the Perceptron classifier to 90% by the Random Forest classifier.Using only the ten best features, the mean correct classification score increased to 95%. CONCLUSION This pilot study provides proof-of-principle that a machine learning approach to analyzing transcripts of interactions between neurologists and patients describing memory problems can distinguish people with neurodegenerative dementia from people with FMD.

Baseline characteristics, analysis plan and report on feasibility for the Prevention Of Decline in Cognition After Stroke Trial (PODCAST)

BackgroundA common complication after stroke is development of cognitive impairment and dementia. However, effective strategies for reducing the risk of developing these problems remain undefined. Potential strategies include intensive lowering of blood pressure (BP) and/or lipids. This paper summarises the baseline characteristics, statistical analysis plan and feasibility of a randomised control trial of blood pressure and lipid lowering in patients post-stroke with the primary objective of reducing cognitive impairment and dementia.MethodsThe Prevention Of Decline in Cognition After Stroke Trial (PODCAST) was a multi-centre prospective randomised open-label blinded-endpoint controlled partial-factorial internal pilot trial running in secondary and primary care. Participants without dementia were enrolled 3–7 months post ischaemic stroke or spontaneous intracerebral haemorrhage, and randomised to intensive versus guideline BP lowering (target systolic BP <125 mmHg versus <140 mmHg); patients with ischaemic stroke were also randomised to intensive or guideline lipid lowering (target LDL cholesterol <1.4 mmol/L versus <3 mmol/L). The primary outcome was the Addenbrooke’s Cognitive Examination-Revised; a key secondary outcome was to assess feasibility of performing a large trial of one or both interventions. Data are number (%) or mean (standard deviation). The trial was planned to last for 8 years with follow-up between 1 and 8 years. The plan for reporting the main results is included as Additional file 2.Results83 patients (of a planned 600) were recruited from 19 UK sites between 7 October 2010 and 31 January 2014. Delays, due to difficulties in the provision of excess treatment costs and to complexity of follow-up, led to few centres taking part and a much lower recruitment rate than planned. Patient characteristics at baseline were: age 74 (SD 7) years, male 64 (77 %), index stroke ischaemic 77 (93 %), stroke onset to randomisation 4.5 [SD 1.3] months, Addenbrooke’s Cognitive Examination-Revised 86 (of 100, SD 8), Montreal Cognitive Assessment 24 (of 30, SD 3), BP 147/82 (SD 19/11) mmHg, total cholesterol 4.0 (SD 0.8) mmol/L and LDL cholesterol 2.0 (SD 0.7) mmol/L, modified Rankin Scale 1.1 (SD 0.8).ConclusionLimited recruitment suggests that a large trial is not feasible using the current protocol. The effects of the interventions on BP, lipids, and cognition will be reported in the main publication.Trial registrationISRCTN85562386 registered on 23 September 2009