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Key Points The calpains are a family of proteases with biologically vital functions. However, the mechanistic features of calpains are largely unknown. Calpains have been identified as potential therapeutic targets for various types of diseases, including neurodegenerative and cardiovascular disorders, ophthalmic diseases and cancer. Many disease phenotypes are ameliorated by calpain inhibition, and some calpain inhibitors have entered clinical trials. Many calpain orthologues in parasites or microorganisms are responsible for the pathogenicity and viability of the organism; thus, targeting these calpains is a promising approach for combatting infectious diseases. Some calpain gene defects resulting in loss of calpain activity are pathologically implicated in human disease. Therefore, in addition to therapies that inhibit calpain activity, developing strategies that compensate for calpain loss are an important goal. The development of inhibitors with improved efficiency and specificity for calpains is a critical future research direction. Unveiling the physiological functions of calpains at the molecular level is a key challenge. The calpain family of proteases are involved in numerous physiological and pathological processes. Here, Sorimachi and colleagues provide an overview of the calpain superfamily and calpain-related disorders, assess the various emerging approaches for therapeutically targeting calpains and highlight agents currently in clinical trials. Calpains are a family of proteases that were scientifically recognized earlier than proteasomes and caspases, but remain enigmatic. However, they are known to participate in a multitude of physiological and pathological processes, performing 'limited proteolysis' whereby they do not destroy but rather modulate the functions of their substrates. Calpains are therefore referred to as 'modulator proteases'. Multidisciplinary research on calpains has begun to elucidate their involvement in pathophysiological mechanisms. Therapeutic strategies targeting malfunctions of calpains have been developed, driven primarily by improvements in the specificity and bioavailability of calpain inhibitors. Here, we review the calpain superfamily and calpain-related disorders, and discuss emerging calpain-targeted therapeutic strategies.

Like a computer, the brain needs a reliable source of power, which is provided as oxygen and glucose in the blood. However, in many neurological disorders this energy supply is disrupted. Brain blood flow is controlled by adjustment of the diameters of the vessels supplying the blood. Nortley et al. found that, both in humans developing Alzheimer's disease (AD) and in a mouse model of AD, brain capillaries become squeezed by pericytes (see the Perspective by Liesz). By defining the underlying mechanism, they suggest potential targets for therapy in early AD. Science , this issue p. eaav9518 ; see also p. 223 Amyloid-β acts on pericytes to reduce brain energy supply in humans with cognitive decline. Cerebral blood flow is reduced early in the onset of Alzheimer’s disease (AD). Because most of the vascular resistance within the brain is in capillaries, this could reflect dysfunction of contractile pericytes on capillary walls. We used live and rapidly fixed biopsied human tissue to establish disease relevance, and rodent experiments to define mechanism. We found that in humans with cognitive decline, amyloid β (Aβ) constricts brain capillaries at pericyte locations. This was caused by Aβ generating reactive oxygen species, which evoked the release of endothelin-1 (ET) that activated pericyte ET A receptors. Capillary, but not arteriole, constriction also occurred in vivo in a mouse model of AD. Thus, inhibiting the capillary constriction caused by Aβ could potentially reduce energy lack and neurodegeneration in AD.

Background Recent studies suggest that microglia contribute to tau pathology progression in Alzheimer’s disease. Amyloid plaque accumulation transforms microglia, the primary innate immune cells in the brain, into neurodegenerative microglia (MGnD), which exhibit enhanced phagocytosis of plaques, apoptotic neurons and dystrophic neurites containing aggregated and phosphorylated tau (p-tau). It remains unclear how microglia promote disease progression while actively phagocytosing pathological proteins, therefore ameliorating pathology. Methods Adeno-associated virus expressing P301L tau mutant (AAV-P301L-tau) was stereotaxically injected into the medial entorhinal cortex (MEC) in C57BL/6 (WT) and humanized APP mutant knock-in homozygote ( App NL-G-F ) mice at 5 months of age. Mice were fed either chow containing a colony stimulating factor-1 receptor inhibitor (PLX5622) or control chow from 4 to 6 months of age to test the effect of microglia depletion. Animals were tested at 6 months of age for immunofluorescence, biochemistry, and FACS of microglia. In order to monitor microglial extracellular vesicle secretion in vivo, a novel lentiviral EV reporter system was engineered to express mEmerald-CD9 (mE-CD9) specifically in microglia, which was injected into the same region of MEC. Results Expressing P301L tau mutant in the MEC induced tau propagation to the granule cell layer of the hippocampal dentate gyrus, which was significantly exacerbated in App NL-G-F mice compared to WT control mice. Administration of PLX5622 depleted nearly all microglia in mouse brains and dramatically reduced propagation of p-tau in WT and to a greater extent in App NL-G-F mice, although it increased plaque burden and plaque-associated p-tau + dystrophic neurites. Plaque-associated MGnD microglia strongly expressed an EV marker, tumor susceptibility gene 101, indicative of heightened synthesis of EVs. Intracortical injection of mE-CD9 lentivirus successfully induced microglia-specific expression of mE-CD9 + EV particles, which were significantly enhanced in Mac2 + MGnD microglia compared to Mac2 − homeostatic microglia. Finally, consecutive intracortical injection of mE-CD9 lentivirus and AAV-P301L-tau into App NL-G-F mice revealed encapsulation of p-tau in microglia-specific mE-CD9 + EVs as determined by super-resolution microscopy and immuno-electron microscopy. Discussion Our findings suggest that MGnD microglia hyper-secrete p-tau + EVs while compacting Aβ plaques and clearing NP tau, which we propose as a novel mechanistic link between amyloid plaque deposition and exacerbation of tau propagation in App NL-G-F mice.

To understand the molecular processes that link Aβ amyloidosis, tauopathy and neurodegeneration, we screened for tau-interacting proteins by immunoprecipitation/LC-MS. We identified the carboxy-terminal PDZ ligand of nNOS (CAPON) as a novel tau-binding protein. CAPON is an adaptor protein of neuronal nitric oxide synthase (nNOS), and activated by the N-methyl-D-aspartate receptor. We observed accumulation of CAPON in the hippocampal pyramidal cell layer in the App NL-G-F -knock-in (KI) brain. To investigate the effect of CAPON accumulation on Alzheimer’s disease (AD) pathogenesis, CAPON was overexpressed in the brain of App NL-G-F mice crossbred with MAPT (human tau)-KI mice. This produced significant hippocampal atrophy and caspase3-dependent neuronal cell death in the CAPON-expressing hippocampus, suggesting that CAPON accumulation increases neurodegeneration. CAPON expression also induced significantly higher levels of phosphorylated, oligomerized and insoluble tau. In contrast, CAPON deficiency ameliorated the AD-related pathological phenotypes in tauopathy model. These findings suggest that CAPON could be a druggable AD target. To understand the molecular processes that link Aβ amyloidosis, tauopathy and neurodegeneration, the authors screened for tau-interacting proteins. They demonstrated that a novel tau binding protein CAPON accelerates tau pathology and neuronal cell death in an Alzheimer’s disease mouse model.

Microglia-mediated neuroinflammation has been implicated in the pathogenesis of Alzheimer’s disease (AD). Although microglia in aging and neurodegenerative disease model mice show a loss of homeostatic phenotype and activation of disease-associated microglia (DAM), a correlation between those phenotypes and the degree of neuronal cell loss has not been clarified. In this study, we performed RNA sequencing of microglia isolated from three representative neurodegenerative mouse models, App NL - G - F/NL - G - F with amyloid pathology, rTg4510 with tauopathy, and SOD1 G93A with motor neuron disease by magnetic activated cell sorting. In parallel, gene expression patterns of the human precuneus with early Alzheimer’s change (n = 11) and control brain (n = 14) were also analyzed by RNA sequencing. We found that a substantial reduction of homeostatic microglial genes in rTg4510 and SOD1 G93A microglia, whereas DAM genes were uniformly upregulated in all mouse models. The reduction of homeostatic microglial genes was correlated with the degree of neuronal cell loss. In human precuneus with early AD pathology, reduced expression of genes related to microglia- and oligodendrocyte-specific markers was observed, although the expression of DAM genes was not upregulated. Our results implicate a loss of homeostatic microglial function in the progression of AD and other neurodegenerative diseases. Moreover, analyses of human precuneus also suggest loss of microglia and oligodendrocyte functions induced by early amyloid pathology in human.

Many mouse models of Alzheimer's disease (AD) rely on overexpression of amyloid precursor (APP) transgenes, which makes it difficult to tease out which effects are truly disease-relevant and which are induced by the overexpression. In this study, the authors describe several new knock-in AD model mice that express mutant APP at near physiological levels. Experimental studies of Alzheimer's disease have largely depended on transgenic mice overexpressing amyloid precursor protein (APP). These mice, however, suffer from artificial phenotypes because, in addition to amyloid β peptide (Aβ), they overproduce other APP fragments. We generated knock-in mice that harbor Swedish and Beyreuther/Iberian mutations with and without the Arctic mutation in the APP gene. The mice showed typical Aβ pathology, neuroinflammation and memory impairment in an age-dependent manner.

Alzheimer’s disease (AD) is characterized by β-amyloid plaques and intraneuronal τ aggregation usually associated with cerebral amyloid angiopathy (CAA). Both β-amyloid plaques and CAA deposits contain fibrillar aggregates of the amyloid β-peptide (Aβ). Aβ plaques and CAA develop first in neocortical areas of preclinical AD patients and, then, expand in a characteristic sequence into further brain regions with end-stage pathology in symptomatic AD patients. Aβ aggregates are not restricted to amyloid plaques and CAA. Soluble and several types of insoluble non-plaque- and non-CAA-associated Aβ aggregates have been described. Amyloid fibrils are products of a complex self-assembly process that involves different types of transient intermediates. Amongst these intermediate species are protofibrils and oligomers. Different variants of Aβ peptides may result from alternative processing or from mutations that lead to rare forms of familial AD. These variants can exhibit different self-assembly and aggregation properties. In addition, several post-translational modifications of Aβ have been described that result, for example, in the production of N-terminal truncated Aβ with pyroglutamate modification at position 3 (Aβ N3pE ) or of Aβ phosphorylated at serine 8 (pSer8Aβ). Both Aβ N3pE and pSer8Aβ show enhanced aggregation into oligomers and fibrils. However, the earliest detectable soluble and insoluble Aβ aggregates in the human brain exhibit non-modified Aβ, whereas Aβ N3pE and pSer8Aβ are detected in later stages. This finding indicates the existence of different biochemical stages of Aβ aggregate maturation with pSer8Aβ being related mainly to cases with symptomatic AD. The conversion from preclinical to symptomatic AD could thereby be related to combined effects of increased Aβ concentration, maturation of aggregates and spread of deposits into additional brain regions. Thus, the inhibition of Aβ aggregation and maturation before entering the symptomatic stage of the disease as indicated by the accumulation of pSer8Aβ may represent an attractive treatment strategy for preventing disease progression.

Alzheimer’s disease (AD) is associated with progressive neuronal degeneration as amyloid-beta (Aβ) and tau proteins accumulate in the brain. Glial cells were recently reported to play an important role in the development of AD. However, little is known about the role of oligodendrocytes in AD pathogenesis. Here, we describe a disease-associated subpopulation of oligodendrocytes that is present during progression of AD-like pathology in the male App NL-G-F and male 5xFAD AD mouse brains and in postmortem AD human brains using single-cell RNA sequencing analysis. Aberrant Erk1/2 signaling was found to be associated with the activation of disease-associated oligodendrocytes (DAOs) in male App NL-G-F mouse brains. Notably, inhibition of Erk1/2 signaling in DAOs rescued impaired axonal myelination and ameliorated Aβ-associated pathologies and cognitive decline in the male App NL-G-F AD mouse model. Oligodendrocytes have been increasingly shown to be involved in Alzheimer’s disease (AD). Here, the authors perform single-cell RNA-sequencing on APP NL-G-F mice and describe a disease-associated oligodendrocyte (DAO) population. They find inhibition of Erk1/2 signaling in DAOs rescues impaired axonal myelination and cognitive decline in an AD mouse model.

Microglial dysfunction is a key pathological feature of Alzheimer's disease (AD), but little is known about proteome-wide changes in microglia during the course of AD and their functional consequences. Here, we performed an in-depth and time-resolved proteomic characterization of microglia in two mouse models of amyloid β (Aβ) pathology, the overexpression APPPS1 and the knock-in APP-NL-G-F (APP-KI) model. We identified a large panel of Microglial Aβ Response Proteins (MARPs) that reflect heterogeneity of microglial alterations during early, middle and advanced stages of Aβ deposition and occur earlier in the APPPS1 mice. Strikingly, the kinetic differences in proteomic profiles correlated with the presence of fibrillar Aβ, rather than dystrophic neurites, suggesting that fibrillar Aβ may trigger the AD-associated microglial phenotype and the observed functional decline. The identified microglial proteomic fingerprints of AD provide a valuable resource for functional studies of novel molecular targets and potential biomarkers for monitoring AD progression or therapeutic efficacy. Alzheimer’s disease is a progressive, irreversible brain disorder. Patients with Alzheimer’s have problems with memory and other mental skills, which lead to more severe cognitive decline and, eventually, premature death. This is due to increasing numbers of nerve cells in the brain dying over time. A distinctive feature of Alzheimer’s is the abnormally high accumulation of a protein called amyloid-β, which forms distinctive clumps in the brain termed ‘plaques’. The brain has a type of cells called the microglia that identify infections, toxic material and damaged cells, and prevent these from building up by clearing them away. In Alzheimer’s disease, however, the microglia do not work properly, which is thought to contribute to the accumulation of amyloid-β plaques. This means that people with mutations in the genes important for the microglia activity are also at higher risk of developing the disease. Although problems with the microglia play an important role in Alzheimer’s, researchers still do not fully understand why microglia stop working in the first place. It is also not known exactly when and how the microglia change as Alzheimer’s disease progresses. To unravel this mystery, Sebastian Monasor, Müller et al. carried out a detailed study of the molecular ‘fingerprints’ of microglia at each key stage of Alzheimer’s disease. The experiments used microglia cells from two different strains of genetically altered mice, both of which develop the hallmarks of Alzheimer’s disease, including amyloid-β plaques, at similar rates. Analysis of the proteins in microglia cells from both strains revealed distinctive, large-scale changes corresponding to successive stages of the disease – reflecting the gradual accumulation of plaques. Obvious defects in microglia function also appeared soon after plaques started to build up. Microscopy imaging of the brain tissue showed that although amyloid-β plaques appeared at the same time, they looked different in each mouse strain. In one, plaques were more compact, while in the other, plaques appeared ‘fluffier’, like cotton wool. In mice with more compacted plaques, microglia recognized the plaques earlier and stopped working sooner, suggesting that plaque structure and microglia defects could be linked. These results shed new light on the role of microglia and their changing protein ‘signals’ during the different stages of Alzheimer’s disease. In the future, this information could help identify people at risk for the disease, so that they can be treated as soon as possible, and to design new therapies to make microglia work again.

Brain microglia and border-associated macrophages (BAMs) display distinct spatial, developmental, and phenotypic features. Although at steady state, the origins of distinct brain macrophages are well-documented, the dynamics of their replenishment in neurodegenerative disorders remain elusive, particularly for activated CD11c + microglia and BAMs. In this study, we conducted a comprehensive fate-mapping analysis of murine microglia and BAMs and their turnover kinetics during Alzheimer’s disease (AD) progression. We used a novel inducible AD mouse model to investigate the contribution of bone marrow (BM) cells to the pool of fetal-derived brain macrophages during the development of AD. We demonstrated that microglia remain a remarkably stable embryonic-derived population even during the progression of AD pathology, indicating that neither parenchymal macrophage subpopulation originates from, nor is replenished by, BM-derived cells. At the border-associated brain regions, bona fide CD206 + BAMs are minimally replaced by BM-derived cells, and their turnover rates are not accelerated by AD. In contrast, all other myeloid cells are swiftly replenished by BM progenitors. This information further elucidates the turnover kinetics of these cells not only at steady state, but also in neurodegenerative diseases, which is crucial for identifying potential novel therapeutic targets.