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Heterozygous gain-of-kinase function variants in LRRK2 (leucine-rich repeat kinase 2) cause 1–2% of all cases of Parkinson’s disease (PD) albeit with incomplete and age-dependent penetrance. All pathogenic LRRK2 mutations reside within the two catalytic domains of LRRK2—either in its kinase domain (e.g. G2019S) with modest effect or its ROC-COR GTPase domain (e.g. R1441G/H) with large effect on LRRK2 kinase activity. We have previously reported assays to interrogate LRRK2 kinase pathway activity in human bio-samples measuring phosphorylation of its endogenous substrate Rab10, that mirrors LRRK2 kinase activation status. Here, we isolated neutrophils from fresh peripheral blood from 101 participants including 42 LRRK2 mutation carriers (21 with the G2019S and 21 with the R1441G mutations), 27 patients with idiopathic PD, and 32 controls. Using a dual approach, LRRK2 dependent Rab10 phosphorylation at Threonine 73 (pRab10Thr73) was measured by quantitative multiplexed immunoblotting for pRab10Thr73/total Rab10 as well as targeted mass-spectrometry for absolute pRab10Thr73 occupancy. We found a significant over fourfold increase in pRab10Thr73 phosphorylation in carriers of the LRRK2 R1441G mutation irrespective of clinical disease status. The effect of the LRRK2 G2019S mutation did not reach statistical significance. Furthermore, we show that LRRK2 phosphorylation at Serine 935 is not a marker for LRRK2 kinase activity in human neutrophils. When analysing pRab10Thr73 phosphorylation in post-mortem brain samples, we observed overall high variability irrespective of clinical and LRRK2 mutation status and attributed this mainly to the adverse effect of the peri- and post-mortem period on the stability of posttranslational modifications such as protein phosphorylation. Overall, in vivo LRRK2 dependent pRab10Thr73 phosphorylation in human peripheral blood neutrophils is a specific, robust and promising biomarker for significant LRRK2 kinase hyperactivation, as with the LRRK2 R1441G mutation. Additional readouts and/or assays may be needed to increase sensitivity to detect modest LRRK2 kinase activation, as with the LRRK2 G2019S mutation. Our assays could be useful for patient stratification and target engagement studies for LRRK2 kinase inhibitors.

Corpora amylacea (CA) in the human brain are polyglucosan bodies that accumulate residual substances originated from aging and both neurodegenerative and infectious processes. These structures, which act as waste containers, are released from the brain to the cerebrospinal fluid, reach the cervical lymph nodes via the meningeal lymphatic system and may be phagocytosed by macrophages. Recent studies indicate that CA present certain neoepitopes (NEs) that can be recognized by natural antibodies of the IgM class, and although evidence of different kinds suggests that these NEs may be formed by carbohydrate structures, their precise nature is unknown. Here, we adapted standard techniques to examine this question. We observed that the preadsorption of IgMs with specific carbohydrates has inhibitory effects on the interaction between IgMs and CA, and found that the digestion of CA proteins had no effect on this interaction. These findings point to the carbohydrate nature of the NEs located in CA. Moreover, the present study indicates that, in vitro , the binding between certain natural IgMs and certain epitopes may be disrupted by certain monosaccharides. We wonder, therefore, whether these inhibitions may also occur in vivo . Further studies should now be carried out to assess the possible in vivo effect of glycemia on the reactivity of natural IgMs and, by extension, on natural immunity.

Background Corpora amylacea of human brain, recently renamed as wasteosomes, are granular structures that appear during aging and also accumulate in specific areas of the brain in neurodegenerative conditions. Acting as waste containers, wasteosomes are formed by polyglucosan aggregates that entrap and isolate toxic and waste substances of different origins. They are expelled from the brain to the cerebrospinal fluid (CSF), and can be phagocytosed by macrophages. In the present study, we analyze the phagocytosis of wasteosomes and the mechanisms involved in this process. Accordingly, we purified wasteosomes from post-mortem extracted human CSF and incubated them with THP-1 macrophages. Immunofluorescence staining and time-lapse recording techniques were performed to evaluate the phagocytosis. We also immunostained human hippocampal sections to study possible interactions between wasteosomes and macrophages at central nervous system interfaces. Results We observed that the wasteosomes obtained from post-mortem extracted CSF are opsonized by MBL and the C3b complement protein. Moreover, we observed that CD206 and CD35 receptors may be involved in the phagocytosis of these wasteosomes by THP-1 macrophages. Once phagocytosed, wasteosomes become degraded and some of the resulting fractions can be exposed on the surface of macrophages and interchanged between different macrophages. However, brain tissue studies show that, in physiological conditions, CD206 but not CD35 receptors may be involved in the phagocytosis of wasteosomes. Conclusions The present study indicates that macrophages have the machinery required to process and degrade wasteosomes, and that macrophages can interact in different ways with wasteosomes. In physiological conditions, the main mechanism involve CD206 receptors and M2 macrophages, which trigger the phagocytosis of wasteosomes without inducing inflammatory responses, thus avoiding tissue damage. However, altered wasteosomes like those obtained from post-mortem extracted CSF, which may exhibit waste elements, become opsonized by MBL and C3b, and so CD35 receptors constitute another possible mechanism of phagocytosis, leading in this case to inflammatory responses.

Background More than 40 pathogenic heterozygous PRNP mutations causing inherited prion diseases have been identified to date. Recessive inherited prion disease has not been described to date. Methods We describe the clinical and neuropathological data of inherited early-onset prion disease caused by the rare PRNP homozygous mutation R136S. In vitro PrP Sc propagation studies were performed using recombinant-adapted protein misfolding cyclic amplification technique. Brain material from two R136S homozygous patients was intracranially inoculated in TgMet129 and TgVal129 transgenic mice to assess the transmissibility of this rare inherited form of prion disease. Results The index case presented symptoms of early-onset dementia beginning at the age of 49 and died at the age of 53. Neuropathological evaluation of the proband revealed abundant multicentric PrP plaques and Western blotting revealed a ~ 8 kDa protease-resistant, unglycosylated PrP Sc fragment, consistent with a Gerstmann-Sträussler-Scheinker phenotype. Her youngest sibling suffered from progressive cognitive decline, motor impairment, and myoclonus with onset in her late 30s and died at the age of 48. Genetic analysis revealed the presence of the R136S mutation in homozygosis in the two affected subjects linked to homozygous methionine at codon 129. One sibling carrying the heterozygous R136S mutation, linked to homozygous methionine at codon 129, is still asymptomatic at the age of 74. The inoculation of human brain homogenates from our index case and an independent case from a Portuguese family with the same mutation in transgenic mice expressing human PrP and in vitro propagation of PrP Sc studies failed to show disease transmissibility. Conclusion In conclusion, biallelic R136S substitution is a rare variant that produces inherited early-onset human prion disease with a Gerstmann-Sträussler-Scheinker neuropathological and molecular signature. Even if the R136S variant is predicted to be “probably damaging”, heterozygous carriers are protected, at least from an early onset providing evidence for a potentially recessive pattern of inheritance in human prion diseases.

Corpora amylacea (CA) in the human brain are granular bodies formed by polyglucosan aggregates that amass waste products of different origins. They are generated by astrocytes, mainly during aging and neurodegenerative conditions, and are located predominantly in periventricular and subpial regions. This study shows that CA are released from these regions to the cerebrospinal fluid and are present in the cervical lymph nodes, into which cerebrospinal fluid drains through the meningeal lymphatic system. We also show that CA can be phagocytosed by macrophages. We conclude that CA can act as containers that remove waste products from the brain and may be involved in a mechanism that cleans the brain. Moreover, we postulate that CA may contribute in some autoimmune brain diseases, exporting brain substances that interact with the immune system, and hypothesize that CA may contain brain markers that may aid in the diagnosis of certain brain diseases.

Cognitive impairment and behavioral changes in amyotrophic lateral sclerosis (ALS) are now recognized as part of the disease. Whether it is solely related to the extent of TDP‐43 pathology is currently unclear. We aim to evaluate the influence of age, genetics, neuropathological features, and concomitant pathologies on cognitive impairment in ALS patients. We analyzed a postmortem series of 104 ALS patients and retrospectively reviewed clinical and neuropathological data. We assessed the burden and extent of concomitant pathologies, the role of APOE ε4 and mutations, and correlated these findings with cognitive status. We performed a logistic regression model to identify which pathologies are related to cognitive impairment. Cognitive decline was recorded in 38.5% of the subjects. Neuropathological features of frontotemporal lobar degeneration (FTLD) were found in 32.7%, explaining most, but not all, cases with cognitive impairment. Extent of TDP‐43 pathology and the presence of hippocampal sclerosis were associated with cognitive impairment. Mutation carriers presented a higher burden of TDP‐43 pathology and FTLD more frequently than sporadic cases. Most cases (89.4%) presented some degree of concomitant pathologies. The presence of concomitant pathologies was associated with older age at death. FTLD, but also Alzheimer’s disease, were the predominant underlying pathologies explaining the cognitive impairment in ALS patients. In sum, FTLD explained the presence of cognitive decline in most but not all ALS cases, while other non‐FTLD related findings can influence the cognitive status, particularly in older age groups.