Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell-surface immunoreceptor expressed on microglia, osteoclasts, dendritic cells and macrophages. Heterozygous loss-of-function mutations in TREM2, including mutations enhancing shedding form the cell surface, have been associated with myelin/neuronal loss and neuroinflammation in neurodegenerative diseases, such as Alzheimer`s disease and Frontotemporal Dementia. Using the cuprizone model, we investigated the involvement of soluble and cleavage-reduced TREM2 on central myelination processes in cleavage-reduced (TREM2-IPD), soluble-only (TREM2-sol), knockout (TREM2-KO) and wild-type (WT) mice. The TREM2-sol mouse is a new model with selective elimination of plasma membrane TREM2 and a reduced expression of soluble TREM2. In the acute cuprizone model demyelination and remyelination events were reflected by a T2-weighted signal intensity change in magnetic resonance imaging (MRI), most prominently in the external capsule (EC). In contrast to WT and TREM2-IPD, TREM2-sol and TREM2-KO showed an additional increase in MRI signal during the recovery phase. Histological analyses of TREM2-IPD animals revealed no recovery of neuroinflammation as well as of the lysosomal marker LAMP-1 and displayed enhanced cytokine/chemokine levels in the brain. TREM2-sol and, to a much lesser extent, TREM2-KO, however, despite presenting reduced levels of some cytokines/chemokines, showed persistent microgliosis and astrocytosis during recovery, with both homeostatic (TMEM119) as well as activated (LAMP-1) microglia markers increased. This was accompanied, specifically in the EC, by no myelin recovery, with appearance of myelin debris and axonal pathology, while oligodendrocytes recovered. In the chronic model consisting of 12-week cuprizone administration followed by 3-week recovery TREM2-IPD displayed sustained microgliosis and enhanced remyelination in the recovery phase. Taken together, our data suggest that sustained microglia activation led to increased remyelination, whereas microglia without plasma membrane TREM2 and only soluble TREM2 had reduced phagocytic activity despite efficient lysosomal function, as observed in bone marrow-derived macrophages, leading to a dysfunctional phenotype with improper myelin debris removal, lack of remyelination and axonal pathology following cuprizone intoxication.

In vivo susceptibility to energy failure parkinsonism and LRRK2 kinase activity.

The G2019S mutation of LRRK2 represents a risk factor for idiopathic Parkinson's disease. Here, we investigate whether LRRK2 kinase activity regulates susceptibility to the environmental toxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). G2019S knock-in mice (bearing enhanced kinase activity) showed greater nigro-striatal degeneration compared to LRRK2 knock-out, LRRK2 kinase-dead and wild-type mice following subacute MPTP treatment. LRRK2 kinase inhibitors PF-06447475 and MLi-2, tested under preventive or therapeutic treatments, protected against nigral dopamine cell loss in G2019S knock-in mice. MLi-2 also rescued striatal dopaminergic terminal degeneration in both G2019S knock-in and wild-type mice. Immunoblot analysis of LRRK2 Serine935 phosphorylation levels confirmed target engagement of LRRK2 inhibitors. However, MLi-2 abolished phosphoSerine935 levels in the striatum and midbrain of both wild-type and G2019S knock-in mice whereas PF-06447475 partly reduced phosphoSerine935 levels in the midbrain of both genotypes. In vivo and ex vivo uptake of the 18-kDa translocator protein (TSPO) ligand [18F]-VC701 revealed a similar TSPO binding in MPTP-treated wild-type and G2019S knock-in mice which was consistent with an increased GFAP striatal expression as revealed by Real Time PCR. We conclude that LRRK2 G2019S, likely through enhanced kinase activity, confers greater susceptibility to mitochondrial toxin-induced parkinsonism. LRRK2 kinase inhibitors are neuroprotective in this model.

Constitutive silencing of LRRK2 kinase activity leads to early glucocerebrosidase deregulation and late impairment of autophagy in vivo.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease. LRRK2 modulates the autophagy-lysosome pathway (ALP), a clearance process subserving the quality control of cellular proteins and organelles. Since dysfunctional ALP might lead to α-synuclein accumulation and, hence, Parkinson's disease, LRRK2 kinase modulation of ALP, its age-dependence and relation with pSer129 α-synuclein inclusions were investigated in vivo. Striatal ALP markers were analyzed by Western blotting in 3, 12 and 20-month-old LRRK2 G2019S knock-in mice (bearing enhanced kinase activity), LRRK2 knock-out mice, LRRK2 D1994S knock-in (kinase-dead) mice and wild-type controls. The lysosomotropic agent chloroquine was used to investigate the autophagic flux in vivo. Quantitative Real-time PCR was used to quantify the transcript levels of key ALP genes. The activity of the lysosomal enzyme glucocerebrosidase was measured using enzymatic assay. Immunohistochemistry was used to co-localize LC3B puncta with pSer129 α-synuclein inclusion in striatal and nigral neurons. No genotype differences in ALP markers were observed at 3 months. Conversely, increase of LC3-I, p62, LAMP2 and GAPDH levels, decrease of p-mTOR levels and downregulation of mTOR and TFEB expression was observed in 12-month-old kinase-dead mice. The LC3-II/I ratio was reduced following administration of chloroquine, suggesting a defective autophagic flux. G2019S knock-in mice showed LAMP2 accumulation and downregulation of ALP key genes MAP1LC3B, LAMP2, mTOR, TFEB and GBA1. Subacute administration of the LRRK2 kinase inhibitor MLi-2 in wild-type and G2019S knock-in mice did not replicate the pattern of kinase-dead mice. Lysosomal glucocerebrosidase activity was increased in 3 and 12-month-old knock-out and kinase-dead mice. LC3B puncta accumulation and pSer129 α-synuclein inclusions were dissociated in striatal neurons of kinase-dead and G2019S knock-in mice. We conclude that constitutive LRRK2 kinase silencing results in early deregulation of GCase activity followed by late impairment of macroautophagy and chaperone-mediated autophagy.

EBI2 is expressed in glial cells in multiple sclerosis lesions, and its knock-out modulates remyelination in the cuprizone model.

EBI2 receptor regulates the immune system, and in multiple, sclerosis is upregulated in the central nervous system infiltrating lymphocytes. In newborn EBI2-deficient mice, myelin development is delayed, and its persistent antagonism inhibits remyelination in chemically demyelinated organotypic cerebellar slices. We used the cuprizone model of multiple sclerosis to elucidate the role of central nervous system-expressed EBI2 in de- and remyelination. The wild-type and EBI2 knock-out mice were fed 0.2% cuprizone in chow for 5 weeks and allowed to recover on a normal diet for 2 weeks. The data showed less efficient recovery of myelin, attenuated oligodendrocyte loss, fewer astrocytes and increased total cholesterol levels in the EBI2 knock-out mice after recovery. Moreover, the wild-type mice upregulated EBI2 expression after recovery confirming the involvement of EBI2 signalling during recovery from demyelination in the cuprizone model. The pro-inflammatory cytokine levels were at comparable levels in the wild-type and EBI2 knock-out mice, with only minor differences in TNFα and IL1ß levels either at peak or during recovery. The neuroinflammatory signalling molecules, Abl1 kinase and NFКB1 (p105/p50) subunit, were significantly downregulated in the EBI2 knock-out mice at peak of disease. Immunohistochemical investigations of EBI2 receptor distribution in the central nervous system (CNS) cells in multiple sclerosis (MS) brain revealed strong expression of EBI2 in astrocytes and microglia inside the plaques implicating glia-expressed EBI2 in multiple sclerosis pathophysiology. Taken together, these findings demonstrate the involvement of EBI2 signalling in the recovery from demyelination rather than in demyelination and as such warrant further research into the role of EBI2 in remyelination.

Early restoration of parvalbumin interneuron activity prevents memory loss and network hyperexcitability in a mouse model of Alzheimer's disease.

Neuronal network dysfunction is increasingly recognized as an early symptom in Alzheimer's disease (AD) and may provide new entry points for diagnosis and intervention. Here, we show that amyloid-beta-induced hyperexcitability of hippocampal inhibitory parvalbumin (PV) interneurons importantly contributes to neuronal network dysfunction and memory impairment in APP/PS1 mice, a mouse model of increased amyloidosis. We demonstrate that hippocampal PV interneurons become hyperexcitable at ~16 weeks of age, when no changes are observed yet in the intrinsic properties of pyramidal cells. This hyperexcitable state of PV interneurons coincides with increased inhibitory transmission onto hippocampal pyramidal neurons and deficits in spatial learning and memory. We show that treatment aimed at preventing PV interneurons from becoming hyperexcitable is sufficient to restore PV interneuron properties to wild-type levels, reduce inhibitory input onto pyramidal cells, and rescue memory deficits in APP/PS1 mice. Importantly, we demonstrate that early intervention aimed at restoring PV interneuron activity has long-term beneficial effects on memory and hippocampal network activity, and reduces amyloid plaque deposition, a hallmark of AD pathology. Taken together, these findings suggest that early treatment of PV interneuron hyperactivity might be clinically relevant in preventing memory decline and delaying AD progression.

Early Aß reduction prevents progression of cerebral amyloid angiopathy.

OBJECTIVE: Clinical trials targeting ß-amyloid peptides (Aß) for Alzheimer disease (AD) failed for arguable reasons that include selecting the wrong stages of AD pathophysiology or Aß being the wrong target. Targeting Aß to prevent cerebral amyloid angiopathy (CAA) has not been rigorously followed, although the causal role of Aß for CAA and related hemorrhages is undisputed. CAA occurs with normal aging and to various degrees in AD, where its impact and treatment is confounded by the presence of parenchymal Aß deposition. METHODS: APPDutch mice develop CAA in the absence of parenchymal amyloid, mimicking hereditary cerebral hemorrhage with amyloidosis Dutch type (HCHWA-D). Mice were treated with a ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor. We used 3-dimensional ultramicroscopy and immunoassays for visualizing CAA and assessing Aß in cerebrospinal fluid (CSF) and brain. RESULTS: CAA onset in mice was at 22 to 24 months, first in frontal leptomeningeal and superficial cortical vessels followed by vessels penetrating the cortical layers. CSF Aß increased with aging followed by a decrease of both Aß40 and Aß42 upon CAA onset, supporting the idea that combined reduction of CSF Aß40 and Aß42 is a specific biomarker for vascular amyloid. BACE1 inhibitor treatment starting at CAA onset and continuing for 4 months revealed a 90% Aß reduction in CSF and largely prevented CAA progression and associated pathologies. INTERPRETATION: This is the first study showing that Aß reduction at early disease time points largely prevents CAA in the absence of parenchymal amyloid. Our observation provides a preclinical basis for Aß-reducing treatments in patients at risk of CAA and in presymptomatic HCHWA-D. ANN NEUROL 2019;86:561-571.

BACE inhibition-dependent repair of Alzheimer's pathophysiology.

Amyloid-ß (Aß) is thought to play an essential pathogenic role in Alzheimer´s disease (AD). A key enzyme involved in the generation of Aß is the ß-secretase BACE, for which powerful inhibitors have been developed and are currently in use in human clinical trials. However, although BACE inhibition can reduce cerebral Aß levels, whether it also can ameliorate neural circuit and memory impairments remains unclear. Using histochemistry, in vivo Ca2+ imaging, and behavioral analyses in a mouse model of AD, we demonstrate that along with reducing prefibrillary Aß surrounding plaques, the inhibition of BACE activity can rescue neuronal hyperactivity, impaired long-range circuit function, and memory defects. The functional neuronal impairments reappeared after infusion of soluble Aß, mechanistically linking Aß pathology to neuronal and cognitive dysfunction. These data highlight the potential benefits of BACE inhibition for the effective treatment of a wide range of AD-like pathophysiological and cognitive impairments.

G2019S LRRK2 mutation facilitates α-synuclein neuropathology in aged mice.

Fibrillization of α-synuclein is instrumental for the development of Parkinson's disease (PD), thus modulating this process can have profound impact on disease initiation/progression. Here, the impact of the p.G2019S mutation of leucine-rich repeat kinase 2 (LRRK2), which is most frequently associated with familial and sporadic PD, on α-synuclein pathology was investigated. G2019S knock-in mice and wild-type controls were injected with a recombinant adeno-associated viral vector serotype 2/9 (AAV2/9) overexpressing human mutant p.A53T α-synuclein (AAV2/9-hα-syn). Control animals were injected with AAV2/9 carrying green fluorescent protein. Motor behavior, transgene expression, α-syn and pSer129 α-syn load, number of nigral dopamine neurons and density of striatal dopaminergic terminals were evaluated. To investigate the effect of aging, experiments were performed in 3- and 12-month-old mice, evaluated 20 and 12 weeks after virus injection, respectively. hα-syn overexpression induced progressive motor deficits, loss of nigral dopaminergic neurons and striatal terminals, and appearance of proteinase K-resistant aggregates of pSer129 α-syn in both young and old mice. Although no genotype difference was observed in 3-month-old mice, degeneration of nigral dopaminergic neurons was higher in 12-month-old G2019S knock-in mice compared with age-matched wild-type controls (-55% vs -39%, respectively). Consistently, a two-fold higher load of pSer129 α-syn aggregates was found in 12-month-old G2019S knock-in mice. We conclude that G2019S LRRK2 facilitates α-synucleinopathy and degeneration of nigral dopaminergic neurons, and that aging is a major determinant of this effect.

BACE1 inhibition more effectively suppresses initiation than progression of ß-amyloid pathology.

BACE1 is the rate-limiting protease in the production of synaptotoxic ß-amyloid (Aß) species and hence one of the prime drug targets for potential therapy of Alzheimer's disease (AD). However, so far pharmacological BACE1 inhibition failed to rescue the cognitive decline in mild-to-moderate AD patients, which indicates that treatment at the symptomatic stage might be too late. In the current study, chronic in vivo two-photon microscopy was performed in a transgenic AD model to monitor the impact of pharmacological BACE1 inhibition on early ß-amyloid pathology. The longitudinal approach allowed to assess the kinetics of individual plaques and associated presynaptic pathology, before and throughout treatment. BACE1 inhibition could not halt but slow down progressive ß-amyloid deposition and associated synaptic pathology. Notably, the data revealed that the initial process of plaque formation, rather than the subsequent phase of gradual plaque growth, is most sensitive to BACE1 inhibition. This finding of particular susceptibility of plaque formation has profound implications to achieve optimal therapeutic efficacy for the prospective treatment of AD.

Prevention of tau increase in cerebrospinal fluid of APP transgenic mice suggests downstream effect of BACE1 inhibition.

INTRODUCTION: The inhibition of the ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a main therapeutic approach for the treatment of Alzheimer's disease (AD). We previously reported an age-related increase of tau protein in the cerebrospinal fluid (CSF) of amyloid ß (Aß) precursor protein (APP) transgenic mice. METHODS: APP transgenic mice were treated with a potent BACE1 inhibitor. CSF tau and CSF Aß levels were assessed. A novel high-sensitivity tau sandwich immunoassay was developed. RESULTS: We demonstrate that long-term BACE1 inhibition prevents CSF tau increase both in early-depositing APP transgenic mice and APP transgenic mice with moderate Aß pathology. DISCUSSION: Our results demonstrate that BACE1 inhibition not only reduces Aß generation but also downstream AD pathophysiology. The tight correlation between Aß aggregation in brain and CSF tau levels renders CSF tau a valuable marker to predict the effectiveness of BACE1 inhibitors in current clinical trials.

Leucine-rich repeat kinase 2-sensitive Na+/Ca2+ exchanger activity in dendritic cells.

Gene variants of the leucine-rich repeat kinase 2 (LRRK2) are associated with susceptibility to Parkinson's disease (PD). Besides brain and periphery, LRRK2 is expressed in various immune cells including dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. However, the function of LRRK2 in the immune system is still incompletely understood. Here, Ca(2+)-signaling was analyzed in DCs isolated from gene-targeted mice lacking lrrk2 (Lrrk2(-/-)) and their wild-type littermates (Lrrk2(+/+)). According to Western blotting, Lrrk2 was expressed in Lrrk2(+/+) DCs but not in Lrrk2(-/-)DCs. Cytosolic Ca(2+) levels ([Ca(2+)]i) were determined utilizing Fura-2 fluorescence and whole cell currents to decipher electrogenic transport. The increase of [Ca(2+)]i following inhibition of sarcoendoplasmatic Ca(2+)-ATPase with thapsigargin (1 µM) in the absence of extracellular Ca(2+) (Ca(2+)-release) and the increase of [Ca(2+)]i following subsequent readdition of extracellular Ca(2+) (SOCE) were both significantly larger in Lrrk2(-/-) than in Lrrk2(+/+) DCs. The augmented increase of [Ca(2+)]i could have been due to impaired Ca(2+) extrusion by K(+)-independent (NCX) and/or K(+)-dependent (NCKX) Na(+)/Ca(2+)-exchanger activity, which was thus determined from the increase of [Ca(2+)]i, (Δ[Ca(2+)]i), and current following abrupt replacement of Na(+) containing (130 mM) and Ca(2+) free (0 mM) extracellular perfusate by Na(+) free (0 mM) and Ca(2+) containing (2 mM) extracellular perfusate. As a result, both slope and peak of Δ[Ca(2+)]i as well as Na(+)/Ca(2+) exchanger-induced current were significantly lower in Lrrk2(-/-) than in Lrrk2(+/+) DCs. A 6 or 24 hour treatment with the LRRK2 inhibitor GSK2578215A (1 µM) significantly decreased NCX1 and NCKX1 transcript levels, significantly blunted Na(+)/Ca(2+)-exchanger activity, and significantly augmented the increase of [Ca(2+)]i following Ca(2+)-release and SOCE. In conclusion, the present observations disclose a completely novel functional significance of LRRK2, i.e., the up-regulation of Na(+)/Ca(2+) exchanger transcription and activity leading to attenuation of Ca(2+)-signals in DCs.

Genetic and pharmacological evidence that G2019S LRRK2 confers a hyperkinetic phenotype, resistant to motor decline associated with aging.

The leucine-rich repeat kinase 2 mutation G2019S in the kinase-domain is the most common genetic cause of Parkinson's disease. To investigate the impact of the G2019S mutation on motor activity in vivo, a longitudinal phenotyping approach was developed in knock-in (KI) mice bearing this kinase-enhancing mutation. Two cohorts of G2019S KI mice and wild-type littermates (WT) were subjected to behavioral tests, specific for akinesia, bradykinesia and overall gait ability, at different ages (3, 6, 10, 15 and 19months). The motor performance of G2019S KI mice remained stable up to the age of 19months and did not show the typical age-related decline in immobility time and stepping activity of WT. Several lines of evidence suggest that enhanced LRRK2 kinase activity is the main contributor to the observed hyperkinetic phenotype of G2019S KI mice: i) KI mice carrying a LRRK2 kinase-dead mutation (D1994S KD) showed a similar progressive motor decline as WT; ii) two LRRK2 kinase inhibitors, H-1152 and Nov-LRRK2-11, acutely reversed the hyperkinetic phenotype of G2019S KI mice, while being ineffective in WT or D1994S KD animals. LRRK2 target engagement in vivo was further substantiated by reduction of LRRK2 phosphorylation at Ser935 in the striatum and cortex at efficacious doses of Nov-LRRK2-11, and in the striatum at efficacious doses of H-1152. In summary, expression of the G2019S mutation in the mouse LRRK2 gene confers a hyperkinetic phenotype that is resistant to age-related motor decline, likely via enhancement of LRRK2 kinase activity. This study provides an in vivo model to investigate the effects of LRRK2 inhibitors on motor function.

Novel one-step immunoassays to quantify α-synuclein: applications for biomarker development and high-throughput screening.

Familial Parkinson disease (PD) can result from α-synuclein gene multiplication, implicating the reduction of neuronal α-synuclein as a therapeutic target. Moreover, α-synuclein content in human cerebrospinal fluid (CSF) represents a PD biomarker candidate. However, capture-based assays for α-synuclein quantification in CSF (such as by ELISA) have shown discrepancies and have limited suitability for high-throughput screening. Here, we describe two sensitive, in-solution, time-resolved Förster's resonance energy transfer (TR-FRET)-based immunoassays for total and oligomeric α-synuclein quantification. CSF analysis showed strong concordance for total α-synuclein content between two TR-FRET assays and, in agreement with a previously characterized 36 h protocol-based ELISA, demonstrated lower α-synuclein levels in PD donors. Critically, the assay suitability for high-throughput screening of siRNA constructs and small molecules aimed at reducing endogenous α-synuclein levels was established and validated. In a small-scale proof of concept compound screen using 384 well plates, signals ranged from <30 to >120% of the mean of vehicle-treated cells for molecules known to lower and increase cellular α-synuclein, respectively. Furthermore, a reverse genetic screen of a kinase-directed siRNA library identified seven genes that modulated α-synuclein protein levels (five whose knockdown increased and two that decreased cellular α-synuclein protein). This provides critical new biological insight into cellular pathways regulating α-synuclein steady-state expression that may help guide further drug discovery efforts. Moreover, we describe an inherent limitation in current α-synuclein oligomer detection methodology, a finding that will direct improvement of future assay design. Our one-step TR-FRET-based platform for α-synuclein quantification provides a novel platform with superior performance parameters for the rapid screening of large biomarker cohorts and of compound and genetic libraries, both of which are essential to the development of PD therapies.

LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells. Mice expressing a LRRK2 kinase-dead (KD) mutant from the endogenous locus displayed similar early-onset pathophysiological changes in kidney but not lung. KD mutants had dramatically reduced full-length LRRK2 protein levels in the kidney and this genetic effect was mimicked pharmacologically in wild-type mice treated with a LRRK2-selective kinase inhibitor. Knock-in (KI) mice expressing the G2019S PD-associated mutation that increases LRRK2 kinase activity showed none of the LRRK2 protein level and histopathological changes observed in KD and KO mice. The autophagy marker LC3 remained unchanged but kidney mTOR and TCS2 protein levels decreased in KD and increased in KO and KI mice. Unexpectedly, KO and KI mice suffered from diastolic hypertension opposed to normal blood pressure in KD mice. Our findings demonstrate a role for LRRK2 in kidney and lung physiology and further show that LRRK2 kinase function affects LRRK2 protein steady-state levels thereby altering putative scaffold/GTPase activity. These novel aspects of peripheral LRRK2 biology critically impact ongoing attempts to develop LRRK2 selective kinase inhibitors as therapeutics for PD.

Discovery of novel indolinone-based, potent, selective and brain penetrant inhibitors of LRRK2.

Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common genetic cause of Parkinson's disease (PD). The most frequent kinase-enhancing mutation is the G2019S residing in the kinase activation domain. This opens up a promising therapeutic avenue for drug discovery targeting the kinase activity of LRRK2 in PD. Several LRRK2 inhibitors have been reported to date. Here, we report a selective, brain penetrant LRRK2 inhibitor and demonstrate by a competition pulldown assay in vivo target engagement in mice.

Sustained Trem2 stabilization accelerates microglia heterogeneity and Aß pathology in a mouse model of Alzheimer's disease.

TREM2 is a transmembrane protein expressed exclusively in microglia in the brain that regulates inflammatory responses to pathological conditions. Proteolytic cleavage of membrane TREM2 affects microglial function and is associated with Alzheimer's disease, but the consequence of reduced TREM2 proteolytic cleavage has not been determined. Here, we generate a transgenic mouse model of reduced Trem2 shedding (Trem2-Ile-Pro-Asp [IPD]) through amino-acid substitution of an ADAM-protease recognition site. We show that Trem2-IPD mice display increased Trem2 cell-surface-receptor load, survival, and function in myeloid cells. Using single-cell transcriptomic profiling of mouse cortex, we show that sustained Trem2 stabilization induces a shift of fate in microglial maturation and accelerates microglial responses to Aß pathology in a mouse model of Alzheimer's disease. Our data indicate that reduction of Trem2 proteolytic cleavage aggravates neuroinflammation during the course of Alzheimer's disease pathology, suggesting that TREM2 shedding is a critical regulator of microglial activity in pathological states.

Microglial inclusions and neurofilament light chain release follow neuronal α-synuclein lesions in long-term brain slice cultures.

BACKGROUND: Proteopathic brain lesions are a hallmark of many age-related neurodegenerative diseases including synucleinopathies and develop at least a decade before the onset of clinical symptoms. Thus, understanding of the initiation and propagation of such lesions is key for developing therapeutics to delay or halt disease progression. METHODS: Alpha-synuclein (αS) inclusions were induced in long-term murine and human slice cultures by seeded aggregation. An αS seed-recognizing human antibody was tested for blocking seeding and/or spreading of the αS lesions. Release of neurofilament light chain (NfL) into the culture medium was assessed. RESULTS: To study initial stages of α-synucleinopathies, we induced αS inclusions in murine hippocampal slice cultures by seeded aggregation. Induction of αS inclusions in neurons was apparent as early as 1week post-seeding, followed by the occurrence of microglial inclusions in vicinity of the neuronal lesions at 2-3 weeks. The amount of αS inclusions was dependent on the type of αS seed and on the culture's genetic background (wildtype vs A53T-αS genotype). Formation of αS inclusions could be monitored by neurofilament light chain protein release into the culture medium, a fluid biomarker of neurodegeneration commonly used in clinical settings. Local microinjection of αS seeds resulted in spreading of αS inclusions to neuronally connected hippocampal subregions, and seeding and spreading could be inhibited by an αS seed-recognizing human antibody. We then applied parameters of the murine cultures to surgical resection-derived adult human long-term neocortical slice cultures from 22 to 61-year-old donors. Similarly, in these human slice cultures, proof-of-principle induction of αS lesions was achieved at 1week post-seeding in combination with viral A53T-αS expressions. CONCLUSION: The successful translation of these brain cultures from mouse to human with the first reported induction of human αS lesions in a true adult human brain environment underlines the potential of this model to study proteopathic lesions in intact mouse and now even aged human brain environments.

Prominent microglial inclusions in transgenic mouse models of α-synucleinopathy that are distinct from neuronal lesions.

Alpha-synucleinopathies are a group of progressive neurodegenerative disorders, characterized by intracellular deposits of aggregated α-synuclein (αS). The clinical heterogeneity of these diseases is thought to be attributed to conformers (or strains) of αS but the contribution of inclusions in various cell types is unclear. The aim of the present work was to study αS conformers among different transgenic (TG) mouse models of α-synucleinopathies. To this end, four different TG mouse models were studied (Prnp-h[A53T]αS; Thy1-h[A53T]αS; Thy1-h[A30P]αS; Thy1-mαS) that overexpress human or murine αS and differed in their age-of-symptom onset and subsequent disease progression. Postmortem analysis of end-stage brains revealed robust neuronal αS pathology as evidenced by accumulation of αS serine 129 (p-αS) phosphorylation in the brainstem of all four TG mouse lines. Overall appearance of the pathology was similar and only modest differences were observed among additionally affected brain regions. To study αS conformers in these mice, we used pentameric formyl thiophene acetic acid (pFTAA), a fluorescent dye with amyloid conformation-dependent spectral properties. Unexpectedly, besides the neuronal αS pathology, we also found abundant pFTAA-positive inclusions in microglia of all four TG mouse lines. These microglial inclusions were also positive for Thioflavin S and showed immunoreactivity with antibodies recognizing the N-terminus of αS, but were largely p-αS-negative. In all four lines, spectral pFTAA analysis revealed conformational differences between microglia and neuronal inclusions but not among the different mouse models. Concomitant with neuronal lesions, microglial inclusions were already present at presymptomatic stages and could also be induced by seeded αS aggregation. Although nature and significance of microglial inclusions for human α-synucleinopathies remain to be clarified, the previously overlooked abundance of microglial inclusions in TG mouse models of α-synucleinopathy bears importance for mechanistic and preclinical-translational studies.

The ß-secretase (BACE) inhibitor NB-360 in preclinical models: From amyloid-ß reduction to downstream disease-relevant effects.

Inhibition of ß-secretase 1 (BACE-1; also known as ß-site amyloid precursor protein-cleaving enzyme-1) is a current approach to fight the amyloid-ß (Aß) deposition in the brains of patients with Alzheimer's disease, and a number of BACE-1 inhibitors are being tested in clinical trials. The BACE-1 inhibitor NB-360, although not a clinical compound, turned out to be a valuable pharmacological tool to investigate the effects of BACE-1 inhibition on the deposition of different Aß species in amyloid precursor protein (APP) transgenic mice. Furthermore, chronic animal studies with NB-360 revealed relationships between BACE-1 inhibition, Aß deposition, and Aß-related downstream effects on neuroinflammation, neuronal function, and markers of neurodegeneration. NB-360 effects on the processing of physiological BACE-1 substrates as well as on nonenzymatic BACE-1 functions have been investigated, complementing studies in BACE-1 knockout mice. Because NB-360 is also an inhibitor for BACE-2, nonclinical studies in adult animals revealed physiological effects of BACE-2 inhibition. LINKED ARTICLES: This article is part of a themed section on Therapeutics for Dementia and Alzheimer's Disease: New Directions for Precision Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.18/issuetoc.