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Objective To evaluate the CSF levels of chitinase proteins during the presymptomatic and early symptomatic phases of amyotrophic lateral sclerosis (ALS). Methods CSF samples were obtained from 16 controls, 55 individuals at‐risk for ALS (including 18 carrying a mutation in C9ORF72 , 33 in SOD1 ), 12 ALS patients, and 7 phenoconverters (individuals diagnosed with ALS during follow‐up). At‐risk individuals and phenoconverters were enrolled through the Pre‐fALS study, which includes individuals carrying an ALS‐associated gene mutation without disease manifestations at initial assessment. Longitudinal CSF collections, where possible, took place every 3‐12 months for ALS patients and every 1‐2 years for others. CSF levels of chitotriosidase 1 (CHIT1), chitinase‐3‐like protein 1 (CHI3L1, YKL‐40) and chitinase‐3‐like protein 2 (CHI3L2, YKL‐39) were measured by ELISA, along with CHIT1 activity. Longitudinal changes in at‐risk individuals and phenoconverters were fitted to linear mixed effects models. Results Slowly rising levels of CHIT1 were observed over time in the at‐risk individuals (slope 0.059 log10[CHIT1] per year, P < 0.001). Among phenoconverters, CHIT1 levels and activity rose more sharply (0.403 log10[CHIT1] per year, P = 0.005; 0.260 log10[CHIT1 activity] per year, P = 0.007). Individual levels of both CHI3L1 and CHI3L2 remained relatively stable over time in all participant groups. Interpretation The CHIT1 neuroinflammatory response is a feature of the late presymptomatic to early symptomatic phases of ALS. This study does not suggest a long prodrome of upregulated glial activity in ALS pathogenesis, but strengthens the place of CHIT1 as part of a panel of biomarkers to objectively assess the impact of immune‐modulatory therapeutic interventions in ALS.

The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C‐terminal fragments of the protein TDP‐43. Here, we tested the hypothesis that highly sensitive mass spectrometry with parallel reaction monitoring (MS‐PRM) can generate a high‐resolution map of pathological TDP‐43 peptide ratios to form the basis for quantitation of abnormal C‐terminal TDP‐43 fragment enrichment. Human cortex and spinal cord, microscopically staged for the presence of p‐TDP‐43, p‐tau, alpha‐synuclein, and beta‐amyloid pathology, were biochemically fractionated and analyzed by immunoblot and MS for the detection of full‐length and truncated (disease‐specific) TDP‐43 peptides. This informed the synthesis of heavy isotope‐labeled peptides for absolute quantification of TDP‐43 by MS‐PRM across 16 ALS, 8 Parkinson’s, 8 Alzheimer’s disease, and 8 aged control cases. We confirmed by immunoblot the previously described enrichment of pathological C‐terminal fragments in ALS‐TDP urea fractions. Subsequent MS analysis resolved specific TDP‐43 N‐ and C‐terminal peptides, including a novel N‐terminal truncation site‐specific peptide. Absolute quantification of peptides by MS‐PRM showed an increased C:N‐terminal TDP‐43 peptide ratio in ALS‐TDP brain compared to normal and disease controls. A C:N‐terminal ratio >1.5 discriminated ALS from controls with a sensitivity of 100% (CI 79.6–100) and specificity of 100% (CI 68–100), and from Parkinson’s and Alzheimer’s disease with a sensitivity of 93% (CI 70–100) and specificity of 100% (CI 68–100). N‐terminal truncation site‐specific peptides were increased in ALS in line with C‐terminal fragment enrichment, but were also found in a proportion of Alzheimer cases with normal C:N‐terminal ratio but coexistent limbic TDP‐43 neuropathological changes. In conclusion this is a novel, sensitive, and specific method to quantify the enrichment of pathological TDP‐43 fragments in human brain, which could form the basis for an antibody‐free assay. Our methodology has the potential to help clarify if specific pathological TDP‐43 peptide signatures are associated with primary or secondary TDP‐43 proteinopathies., The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C‐terminal fragments of the protein TDP‐43. Here, we used a high‐resolution map of TDP‐43 peptides to form the basis for absolute quantitation of abnormal C‐terminal TDP‐43 fragment enrichment. The C:N‐terminal peptide ratio is increased in ALS brains and N‐terminal truncation‐specific peptides confirm the pathological cleavage of TDP‐43 in ALS and surprisingly AD.

OBJECTIVE The neurodegenerative disease, amyotrophic lateral sclerosis (ALS), is a heterogeneous clinical syndrome involving multiple molecular pathways. The development of biomarkers for use in therapeutic trials is a priority. We sought to use a high-throughput proteomic method to identify novel biomarkers in individual cerebrospinal fluid (CSF) samples. METHODS Liquid chromatography/tandem mass spectrometry with label-free quantification was used to identify CSF proteins using samples from a well-characterized longitudinal cohort comprising patients with ALS (n = 43), the upper motor neuron variant, primary lateral sclerosis (PLS; n = 6), and cross-sectional healthy (n = 20) and disease controls (Parkinsons' disease, n = 20; ALS mimic disorders, n = 12). RESULTS Three macrophage-derived chitinases showed increased abundance in ALS: chitotriosidase (CHIT1), chitinase-3-like protein 1 (CHI3L1), and chitinase-3-like protein 2 (CHI3L2). Elevated CHI3L1 was common to ALS and PLS, whereas CHIT1 and CHI3L2 levels differed. Chitinase levels correlated with disease progression rate (CHIT1, r = 0.56, p < 0.001; CHI3L1, r = 0.31; p = 0.028; CHI3L2, r = 0.29, p = 0.044). CHIT1, CHI3L1, and CHI3L2 levels correlated with phosphorylated neurofilament heavy chain (pNFH; r = 0.62, p < 0.001; r = 0.49, p < 0.001; r = 0.41, p < 0.001). CHI3L1 levels, but not CHIT1 or CHI3L2, increased over time in those with low initial levels (gradient = 0.005 log abundance units/month, p = 0.001). High CHIT1 was associated with shortened survival (hazard ratio [HR] 2.84; p = 0.009). Inclusion of pNFH in survival models left only an association of pNFH and survival (HR 1.26; p = 0.019). INTERPRETATION Neuroinflammatory mechanisms have been consistently implicated through various experimental paradigms. These results support a key role for macrophage activity in ALS pathogenesis, offering novel target engagement and pharmacodynamic biomarkers for neuroinflammation-focused ALS therapy. Ann Neurol 2018;83:258-268.

Objective Spinal muscular atrophy (SMA) is a major inherited cause of infant death worldwide. It results from mutations in a single, ubiquitously expressed gene (SMN1), with loss of lower motor neurons being the primary pathological signature. Systemic defects have also been reported in SMA patients and animal models. We investigated whether defects associated with the vasculature contribute to motor neuron pathology in SMA. Methods Development and integrity of the capillary bed was examined in skeletal muscle and spinal cord of SMA mice, and muscle biopsies from SMA patients and controls, using quantitative morphometric approaches on immunohistochemically labeled tissue. Pimonidazole hydrochloride–based assays were used to identify functional hypoxia. Results The capillary bed in muscle and spinal cord was normal in presymptomatic SMA mice (postnatal day 1), but failed to match subsequent postnatal development in control littermates. At mid- and late-symptomatic time points, the extent of the vascular architecture observed in two distinct mouse models of SMA was ∼50% of that observed in control animals. Skeletal muscle biopsies from human patients confirmed the presence of developmentally similar, significant vascular depletion in severe SMA. Hypovascularity in SMA mouse spinal cord was accompanied by significant functional hypoxia and defects in the blood–spinal cord barrier. Interpretation Our results indicate that vascular defects are a major feature of severe forms of SMA, present in both mouse models and patients, resulting in functional hypoxia of motor neurons. Thus, abnormal vascular development and resulting hypoxia may contribute to the pathogenesis of SMA. Ann Neurol 2016;79:217–230

Motor neuron disease (MND) is characterised by progressive neurological deterioration and coexistence of upper and lower motor neuron signs. Over the past decade, evidence has emerged of unique pathophysiological processes, including glutamate-mediated excitotoxicity, which has resulted in the development of novel diagnostic investigations and uncovered potential therapeutic targets. Advances in genetics, including the recently discovered C9orf72 gene, have radically changed the pathological mindset, from MND being classified as a neuromuscular disease to one that MND forms a continuum with other primary neurodegenerative disorders, including frontotemporal dementia. The present review will highlight the improvements that have occurred in clinical care, in conjunction with recent scientific developments.

Previous imaging studies that investigated morphometric group differences of subcortical regions outside the substantia nigra between non-demented Parkinson's patients and controls either did not find any significant differences, or reported contradictory results. Here, we performed a comprehensive morphometric analysis of 20 cognitively normal, early-stage PD patients and 19 matched control subjects. In addition to relatively standard analyses of whole-brain grey matter volume and overall regional volumes, we examined subtle localized surface shape differences in striatal and limbic grey matter structures and tested their utility as a diagnostic marker. Voxel-based morphometry and volumetric comparisons did not reveal significant group differences. Shape analysis, on the other hand, demonstrated significant between-group shape differences for the right pallidum. Careful diffusion tractography analysis showed that the affected parts of the pallidum are connected subcortically with the subthalamic nucleus, the pedunculopontine nucleus, and the thalamus and cortically with the frontal lobe. Additionally, microstructural measurements along these pathways, but not along other pallidal connections, were significantly different between the two groups. Vertex-wise linear discriminant analysis, however, revealed limited accuracy of pallidal shape for the discrimination between patients and controls. We conclude that localized disease-related changes in the right pallidum in early Parkinson's disease, undetectable using standard voxel-based morphometry or volumetry, are evident using sensitive shape analysis. However, the subtle nature of these changes makes it unlikely that shape analysis alone will be useful for early diagnosis.

Please cite this paper as: Ioannou C, Talbot K, Ohuma E, Sarris I, Villar J, Conde-Agudelo A, Papageorghiou A. Systematic review of methodology used in ultrasound studies aimed at creating charts of fetal size. BJOG 2012;119:1425–1439. Background Reliable ultrasound charts are necessary for the prenatal assessment of fetal size, yet there is a wide variation of methodologies for the creation of such charts. Objective To evaluate the methodological quality of studies of fetal biometry using a set of predefined quality criteria of study design, statistical analysis and reporting methods. Search strategy Electronic searches in MEDLINE, EMBASE and CINAHL, and references of retrieved articles. Selection criteria Observational studies whose primary aim was to create ultrasound size charts for bi-parietal diameter, head circumference, abdominal circumference and femur length in fetuses from singleton pregnancies. Data collection and analysis Studies were scored against a predefined set of independently agreed methodological criteria and an overall quality score was given to each study. Multiple regression analysis between quality scores and study characteristics was performed. Main results Eighty-three studies met the inclusion criteria. The highest potential for bias was noted in the following fields: ‘Inclusion/exclusion criteria’, as none of the studies defined a rigorous set of antenatal or fetal conditions which should be excluded from analysis; ‘Ultrasound quality control measures’, as no study demonstrated a comprehensive quality assurance strategy; and ‘Sample size calculation’, which was apparent in six studies only. On multiple regression analysis, there was a positive correlation between quality scores and year of publication: quality has improved with time, yet considerable heterogeneity in study methodology is still observed today. Conclusions There is considerable methodological heterogeneity in studies of fetal biometry. Standardisation of methodologies is necessary in order to make correct interpretations and comparisons between different charts. A checklist of recommended methodologies is proposed.

J. Neurochem. (2011) 10.1111/j.1471-4159.2011.07521.x Abstract HspB8, a small heat-shock protein implicated in autophagy, is mutated in patients with distal hereditary motor neuropathy type II (dHMNII). Autophagy is essential for maintaining protein homeostasis in the central nervous system, but its role has not been investigated in peripheral motor neurons. We used a novel, multispectral-imaging flow cytometry assay to measure autophagy in cells. This assay revealed that over-expression of wild-type HspB8 in motor neuron-like NSC34 cells led to an increased co-localisation of autophagosomes with the lysosomes. By contrast, over-expression of mutant HspB8 resulted in autophagosomes that co-localised with protein aggregates but failed to co-localise with the lysosomes. A similar impairment of autophagy could also be demonstrated in peripheral blood mononuclear cells from two dHMNII patients with the HspB8K141E mutation. We conclude that defects in HspB8-mediated autophagy are likely to contribute to dHMNII pathology and their detection in peripheral blood mononuclear cells could be a useful, accessible biomarker for the disease.

Amid the great diversity of neurodegenerative conditions, there is a growing body of evidence that non-somatic (that is, synaptic and distal axonal) compartments of neurones are early and important subcellular sites of pathological change. In this review we discuss experimental data from human patients, animal models and in vitro systems showing that neuromuscular synapses are targeted in different forms of motor neurone disease (MND), including amyotrophic lateral sclerosis and spinal muscular atrophy. We highlight important developments revealing the heterogeneous nature of vulnerability in populations of lower motor units in MND and examine how progress in our understanding of the molecular pathways underlying MND may provide insights into the regulation of synaptic vulnerability and pathology. We conclude that future experiments developing therapeutic approaches specifically targeting neuromuscular synaptic vulnerability are likely to be required to prevent or delay disease onset and progression in human MND patients.