Hsueh Cheng Chiang

Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis

The mixed lineage kinase domain-like protein (MLKL) has recently been identified as a key RIP3 (receptor interacting protein 3) downstream component of tumour necrosis factor (TNF)-induced necroptosis. MLKL is phosphorylated by RIP3 and is recruited to the necrosome through its interaction with RIP3. However, it is still unknown how MLKL mediates TNF-induced necroptosis. Here, we report that MLKL forms a homotrimer through its amino-terminal coiled-coil domain and locates to the cell plasma membrane during TNF-induced necroptosis. By generating different MLKL mutants, we demonstrated that the plasma membrane localization of trimerized MLKL is critical for mediating necroptosis. Importantly, we found that the membrane localization of MLKL is essential for Ca2+ influx, which is an early event of TNF-induced necroptosis. Furthermore, we identified that TRPM7 (transient receptor potential melastatin related 7) is a MLKL downstream target for the mediation of Ca2+ influx and TNF-induced necroptosis. Hence, our study reveals a crucial mechanism of MLKL-mediated TNF-induced necroptosis.

BACE1, a Major Determinant of Selective Vulnerability of the Brain to Amyloid-β Amyloidogenesis, is Essential for Cognitive, Emotional, and Synaptic Functions

A transmembrane aspartyl protease termed β-site APP cleavage enzyme 1 (BACE1) that cleaves the amyloid-β precursor protein (APP), which is abundant in neurons, is required for the generation of amyloid-β (Aβ) peptides implicated in the pathogenesis of Alzheimers disease (AD). We now demonstrate that BACE1, enriched in neurons of the CNS, is a major determinant that predisposes the brain to Aβ amyloidogenesis. The physiologically high levels of BACE1 activity coupled with low levels of BACE2 and α-secretase anti-amyloidogenic activities in neurons is a major contributor to the accumulation of Aβ in the CNS, whereas other organs are spared. Significantly, deletion of BACE1 in APPswe;PS1ΔE9 mice prevents both Aβ deposition and age-associated cognitive abnormalities that occur in this model of Aβ amyloidosis. Moreover, Aβ deposits are sensitive to BACE1 dosage and can be efficiently cleared from the CNS when BACE1 is silenced. However, BACE1 null mice manifest alterations in hippocampal synaptic plasticity as well as in performance on tests of cognition and emotion. Importantly, memory deficits but not emotional alterations in BACE1–/– mice are prevented by coexpressing APPswe;PS1ΔE9 transgenes, indicating that other potential substrates of BACE1 may affect neural circuits related to emotion. Our results establish BACE1 and APP processing pathways as critical for cognitive, emotional, and synaptic functions, and future studies should be alert to potential mechanism-based side effects that may occur with BACE1 inhibitors designed to ameliorate Aβ amyloidosis in AD.

Exocytosis and Endocytosis: Modes, Functions, and Coupling Mechanisms*

Vesicle exocytosis releases content to mediate many biological events, including synaptic transmission essential for brain functions. Following exocytosis, endocytosis is initiated to retrieve exocytosed vesicles within seconds to minutes. Decades of studies in secretory cells reveal three exocytosis modes coupled to three endocytosis modes: (a) full-collapse fusion, in which vesicles collapse into the plasma membrane, followed by classical endocytosis involving membrane invagination and vesicle reformation; (b) kiss-and-run, in which the fusion pore opens and closes; and (c) compound exocytosis, which involves exocytosis of giant vesicles formed via vesicle-vesicle fusion, followed by bulk endocytosis that retrieves giant vesicles. Here we review these exo- and endocytosis modes and their roles in regulating quantal size and synaptic strength, generating synaptic plasticity, maintaining exocytosis, and clearing release sites for vesicle replenishment. Furthermore, we highlight recent progress in understanding how vesicle endocytosis is initiated and is thus coupled to exocytosis. The emerging model is that calcium influx via voltage-dependent calcium channels at the calcium microdomain triggers endocytosis and controls endocytosis rate; calmodulin and synaptotagmin are the calcium sensors; and the exocytosis machinery, including SNARE proteins (synaptobrevin, SNAP25, and syntaxin), is needed to coinitiate endocytosis, likely to control the amount of endocytosis.

Aβ42 Mutants with Different Aggregation Profiles Induce Distinct Pathologies in Drosophila

Aggregation of the amyloid-β-42 (Aβ42) peptide in the brain parenchyma is a pathological hallmark of Alzheimers disease (AD), and the prevention of Aβ aggregation has been proposed as a therapeutic intervention in AD. However, recent reports indicate that Aβ can form several different prefibrillar and fibrillar aggregates and that each aggregate may confer different pathogenic effects, suggesting that manipulation of Aβ42 aggregation may not only quantitatively but also qualitatively modify brain pathology. Here, we compare the pathogenicity of human Aβ42 mutants with differing tendencies to aggregate. We examined the aggregation-prone, EOFAD-related Arctic mutation (Aβ42Arc) and an artificial mutation (Aβ42art) that is known to suppress aggregation and toxicity of Aβ42 in vitro. In the Drosophila brain, Aβ42Arc formed more oligomers and deposits than did wild type Aβ42, while Aβ42art formed fewer oligomers and deposits. The severity of locomotor dysfunction and premature death positively correlated with the aggregation tendencies of Aβ peptides. Surprisingly, however, Aβ42art caused earlier onset of memory defects than Aβ42. More remarkably, each Aβ induced qualitatively different pathologies. Aβ42Arc caused greater neuron loss than did Aβ42, while Aβ42art flies showed the strongest neurite degeneration. This pattern of degeneration coincides with the distribution of Thioflavin S-stained Aβ aggregates: Aβ42Arc formed large deposits in the cell body, Aβ42art accumulated preferentially in the neurites, while Aβ42 accumulated in both locations. Our results demonstrate that manipulation of the aggregation propensity of Aβ42 does not simply change the level of toxicity, but can also result in qualitative shifts in the pathology induced in vivo.

Loss of ALS2 Function Is Insufficient to Trigger Motor Neuron Degeneration in Knock-Out Mice But Predisposes Neurons to Oxidative Stress

Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease, is caused by a selective loss of motor neurons in the CNS. Mutations in the ALS2 gene have been linked to one form of autosomal recessive juvenile onset ALS (ALS2). To investigate the pathogenic mechanisms of ALS2, we generated ALS2 knock-out (ALS2-/-) mice. Although ALS2-/- mice lacked obvious developmental abnormalities, they exhibited age-dependent deficits in motor coordination and motor learning. Moreover, ALS2-/- mice showed a higher anxiety response in the open-field and elevated plus-maze tasks. Although they failed to recapitulate clinical or neuropathological phenotypes consistent with motor neuron disease by 20 months of age, ALS2-/- mice or primary cultured neurons derived from these mice were more susceptible to oxidative stress compared with wild-type controls. These observations suggest that loss of ALS2 function is insufficient to cause major motor deficits or motor neuron degeneration in a mouse model but predisposes neurons to oxidative stress.

Amyotrophic Lateral Sclerosis 2-Deficiency Leads to Neuronal Degeneration in Amyotrophic Lateral Sclerosis through Altered AMPA Receptor Trafficking

Amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disease is caused by a selective loss of motor neurons. One form of juvenile onset autosomal recessive ALS (ALS2) has been linked to the loss of function of the ALS2 gene. The pathogenic mechanism of ALS2-deficiency, however, remains unclear. To further understand the function of alsin that is encoded by the full-length ALS2 gene, we screened proteins interacting with alsin. Here, we report that alsin interacted with glutamate receptor interacting protein 1 (GRIP1) both in vitro and in vivo, and colocalized with GRIP1 in neurons. In support of the physiological interaction between alsin and GRIP1, the subcellular distribution of GRIP1 was altered in ALS2 −/− spinal motor neurons, which correlates with a significant reduction of AMPA-type glutamate receptor subunit 2 (GluR2) at the synaptic/cell surface of ALS2 −/− neurons. The decrease of calcium-impermeable GluR2-containing AMPA receptors at the cell/synaptic surface rendered ALS2 −/− neurons more susceptible to glutamate receptor-mediated neurotoxicity. Our findings reveal a novel function of alsin in AMPA receptor trafficking and provide a novel pathogenic link between ALS2-deficiency and motor neuron degeneration, suggesting a protective role of alsin in maintaining the survival of motor neurons.

Epidermal growth factor receptor is a preferred target for treating Amyloid-β–induced memory loss

Current understanding of amyloid-β (Aβ) metabolism and toxicity provides an extensive list of potential targets for developing drugs for treating Alzheimer’s disease. We took two independent approaches, including synaptic-plasticity–based analysis and behavioral screening of synthetic compounds, for identifying single compounds that are capable of rescuing the Aβ-induced memory loss in both transgenic fruit fly and transgenic mouse models. Two clinically available drugs and three synthetic compounds not only showed positive effects in behavioral tests but also antagonized the Aβ oligomers-induced activation of the epidermal growth factor receptor (EGFR). Such surprising converging outcomes from two parallel approaches lead us to conclude that EGFR is a preferred target for treating Aβ-induced memory loss.

PI3 kinase signaling is involved in Aβ-induced memory loss in Drosophila

Multiple intracellular signals are altered in Alzheimers disease brain tissues, including the PI3K/Akt pathway. However, the pathological relevance of such alterations is poorly understood. In vitro studies yield results that seem to be consistent with the conventional perception in which an up-regulation of the cell survival pathway, PI3K pathway, is protective in Alzheimers disease pathogenesis. The current in vivo genetic approach, however, reveals that inhibition of the PI3K pathway leads to rescuing of the β-amyloid peptide (Aβ)-induced memory loss in the Drosophila brain. We began our inquiry into the molecular basis of this memory loss by studying Aβ42-induced enhancement of long-term depression. We found that long-term depression is restored to a normal level through inhibition of PI3K activity. Aβ42-induced PI3K hyperactivity is directly confirmed by immunostaining of the PI3K phosphorylation targets, phospholipids. Such observations lead to the following demonstration that Aβ42-induced memory loss can be rescued through genetic silencing or pharmacological inhibition of PI3K functions. Our data suggest that Aβ42 stimulates PI3K, which in turn causes memory loss in association with an increase in accumulation of Aβ42 aggregates.

Overexpression of Neprilysin Reduces Alzheimer Amyloid-β42 (Aβ42)-induced Neuron Loss and Intraneuronal Aβ42 Deposits but Causes a Reduction in cAMP-responsive Element-binding Protein-mediated Transcription, Age-dependent Axon Pathology, and Premature Death in Drosophila

The amyloid-β42 (Aβ42) peptide has been suggested to play a causative role in Alzheimer disease (AD). Neprilysin (NEP) is one of the rate-limiting Aβ-degrading enzymes, and its enhancement ameliorates extracellular amyloid pathology, synaptic dysfunction, and memory defects in mouse models of Aβ amyloidosis. In addition to the extracellular Aβ, intraneuronal Aβ42 may contribute to AD pathogenesis. However, the protective effects of neuronal NEP expression on intraneuronal Aβ42 accumulation and neurodegeneration remain elusive. In contrast, sustained NEP activation may be detrimental because NEP can degrade many physiological peptides, but its consequences in the brain are not fully understood. Using transgenic Drosophila expressing human NEP and Aβ42, we demonstrated that NEP efficiently suppressed the formation of intraneuronal Aβ42 deposits and Aβ42-induced neuron loss. However, neuronal NEP overexpression reduced cAMP-responsive element-binding protein-mediated transcription, caused age-dependent axon degeneration, and shortened the life span of the flies. Interestingly, the mRNA levels of endogenous fly NEP genes and phosphoramidon-sensitive NEP activity declined during aging in fly brains, as observed in mammals. Taken together, these data suggest both the protective and detrimental effects of chronically high NEP activity in the brain. Down-regulation of NEP activity in aging brains may be an evolutionarily conserved phenomenon, which could predispose humans to developing late-onset AD.

Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane

Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30–300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging.