Broad activation of the Parkin pathway induces synaptic mitochondrial deficits in early tauopathy.

Mitochondrial defects are a hallmark of early pathophysiology in Alzheimer's disease, with pathologically phosphorylated tau reported to induce mitochondrial toxicity. Mitophagy constitutes a key pathway in mitochondrial quality control by which damaged mitochondria are targeted for autophagy. However, few details are known regarding the intersection of mitophagy and pathologies in tauopathy. Here, by applying biochemical and cell biological approaches including time-lapse confocal imaging in live tauopathy neurons, combined with gene rescue experiments via stereotactic injections of adeno-associated virus particles into tauopathy mouse brains, electrophysiological recordings and behavioural tests, we demonstrate for the first time that mitochondrial distribution deficits at presynaptic terminals are an early pathological feature in tauopathy brains. Furthermore, Parkin-mediated mitophagy is extensively activated in tauopathy neurons, which accelerates mitochondrial Rho GTPase 1 (Miro1) turnover and consequently halts Miro1-mediated mitochondrial anterograde movement towards synaptic terminals. As a result, mitochondrial supply at tauopathy synapses is disrupted, impairing synaptic function. Strikingly, increasing Miro1 levels restores the synaptic mitochondrial population by enhancing mitochondrial anterograde movement and thus reverses tauopathy-associated synaptic failure. In tauopathy mouse brains, overexpression of Miro1 markedly elevates synaptic distribution of mitochondria and protects against synaptic damage and neurodegeneration, thereby counteracting impairments in learning and memory as well as synaptic plasticity. Taken together, our study reveals that activation of the Parkin pathway triggers an unexpected effect-depletion of mitochondria from synaptic terminals, a characteristic feature of early tauopathy. We further provide new mechanistic insights into how parkin activation-enhanced Miro1 degradation and impaired mitochondrial anterograde transport drive tauopathy-linked synaptic pathogenesis and establish a foundation for future investigations into new therapeutic strategies to prevent synaptic deterioration in Alzheimer's disease and other tauopathies.

Mitophagy regulates integrity of mitochondria at synapses and is critical for synaptic maintenance.

Synaptic mitochondria are particularly vulnerable to physiological insults, and defects in synaptic mitochondria are linked to early pathophysiology of Alzheimer's disease (AD). Mitophagy, a cargo-specific autophagy for elimination of dysfunctional mitochondria, constitutes a key quality control mechanism. However, how mitophagy ensures synaptic mitochondrial integrity remains largely unknown. Here, we reveal Rheb and Snapin as key players regulating mitochondrial homeostasis at synapses. Rheb initiates mitophagy to target damaged mitochondria for autophagy, whereas dynein-Snapin-mediated retrograde transport promotes clearance of mitophagosomes from synaptic terminals. We demonstrate that synaptic accumulation of mitophagosomes is a feature in AD-related mutant hAPP mouse brains, which is attributed to increased mitophagy initiation coupled with impaired removal of mitophagosomes from AD synapses due to defective retrograde transport. Furthermore, while deficiency in dynein-Snapin-mediated retrograde transport recapitulates synaptic mitophagy stress and induces synaptic degeneration, elevated Snapin expression attenuates mitochondrial defects and ameliorates synapse loss in AD mouse brains. Taken together, our study provides new insights into mitophagy regulation of synaptic mitochondrial integrity, establishing a foundation for mitigating AD-associated mitochondria deficits and synaptic damage through mitophagy enhancement.

Impaired axonal retrograde trafficking of the retromer complex augments lysosomal deficits in Alzheimer's disease neurons.

Lysosomal proteolysis is essential for the quality control of intracellular components and the maintenance of cellular homeostasis. Lysosomal alterations have been implicated as one of the main cellular defects contributing to the onset and progression of Alzheimer's disease (AD). However, the mechanism underlying lysosomal deficits in AD remains poorly understood. Here, we reveal that lysosomal deficits are attributed to retromer dysfunction induced by altered retromer trafficking in the axon of AD-related mutant human amyloid precursor protein (hAPP) transgenic (Tg) mouse neurons. We demonstrate that retrograde transport of retromer is impaired, leading to its significant reduction in the soma and abnormal retention within late endosomes in distal axons of mutant hAPP neurons. Therefore, retromer-mediated endosome-to-Golgi retrieval of cation-independent mannose-6-phosphate receptors (CI-MPR) in the soma is disrupted in mutant hAPP neurons, causing defects in lysosome biogenesis. Such defects result in protease deficiency in lysosomes and impaired lysosomal proteolysis, as evidenced by aberrant accumulation of sequestered substrates within lysosomes. Intriguingly, enhancement of retrograde transport in mutant hAPP neurons facilitates the trafficking of axonal retromer toward the soma and thus enhances protease transport to lysosomes, thereby restoring lysosomal proteolytic activity. Taken together, our study provides new insights into the regulation of retromer trafficking through retrograde axonal transport to fulfil its function in promoting lysosome biogenesis in the soma, suggesting a potential approach for rescuing lysosomal proteolysis deficits in AD.

Regulation of Synaptic Amyloid-ß Generation through BACE1 Retrograde Transport in a Mouse Model of Alzheimer's Disease.

Amyloid-ß (Aß) peptides play a key role in synaptic damage and memory deficits in the early pathogenesis of Alzheimer's disease (AD). Abnormal accumulation of Aß at nerve terminals leads to synaptic pathology and ultimately to neurodegeneration. ß-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the major neuronal ß-secretase for Aß generation. However, the mechanisms regulating BACE1 distribution in axons and ß cleavage of APP at synapses remain largely unknown. Here, we reveal that dynein-Snapin-mediated retrograde transport regulates BACE1 trafficking in axons and APP processing at presynaptic terminals. BACE1 is predominantly accumulated within late endosomes at the synapses of AD-related mutant human APP (hAPP) transgenic (Tg) mice and patient brains. Defective retrograde transport by genetic ablation of snapin in mice recapitulates late endocytic retention of BACE1 and increased APP processing at presynaptic sites. Conversely, overexpressing Snapin facilitates BACE1 trafficking and reduces synaptic BACE1 accumulation by enhancing the removal of BACE1 from distal AD axons and presynaptic terminals. Moreover, elevated Snapin expression via stereotactic hippocampal injections of adeno-associated virus particles in mutant hAPP Tg mouse brains decreases synaptic Aß levels and ameliorates synapse loss, thus rescuing cognitive impairments associated with hAPP mice. Altogether, our study provides new mechanistic insights into the complex regulation of BACE1 trafficking and presynaptic localization through Snapin-mediated dynein-driven retrograde axonal transport, thereby suggesting a potential approach of modulating Aß levels and attenuating synaptic deficits in AD.SIGNIFICANCE STATEMENT ß-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) trafficking and synaptic localization significantly influence its ß secretase activity and amyloid-ß (Aß) production. In AD brains, BACE1 is accumulated within dystrophic neurites, which is thought to augment Aß-induced synaptotoxicity by Aß overproduction. However, it remains largely unknown whether axonal transport regulates synaptic APP processing. Here, we demonstrate that Snapin-mediated retrograde transport plays a critical role in removing BACE1 from presynaptic terminals toward the soma, thus reducing synaptic Aß production. Adeno-associated virus-mediated Snapin overexpression in the hippocampus of mutant hAPP mice significantly decreases synaptic Aß levels, attenuates synapse loss, and thus rescues cognitive deficits. Our study uncovers a new pathway that controls synaptic APP processing by enhancing axonal BACE1 trafficking, thereby advancing our fundamental knowledge critical for ameliorating Aß-linked synaptic pathology.

Autophagy-mediated Regulation of BACE1 Protein Trafficking and Degradation.

ß-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the major neuronal ß-secretase for amyloid-ß generation and is degraded in lysosomes. The autophagy-lysosomal system plays a key role in the maintenance of cellular homeostasis in neurons. Recent studies established that nascent autophagosomes in distal axons move predominantly in the retrograde direction toward the soma, where mature lysosomes are mainly located. However, it remains unknown whether autophagy plays a critical role in regulation of BACE1 trafficking and degradation. Here, we report that induction of neuronal autophagy enhances BACE1 turnover, which is suppressed by lysosomal inhibition. A significant portion of BACE1 is recruited to the autophagy pathway and co-migrates robustly with autophagic vacuoles along axons. Moreover, we reveal that autophagic vacuole-associated BACE1 is accumulated in the distal axon of Alzheimer's disease-related mutant human APP transgenic neurons and mouse brains. Inducing autophagy in mutant human APP neurons augments autophagic retention of BACE1 in distal axons, leading to enhanced ß-cleavage of APP. This phenotype can be reversed by Snapin-enhanced retrograde transport, which facilitates BACE1 trafficking to lysosomes for degradation. Therefore, our study provides new insights into autophagy-mediated regulation of BACE1 turnover and APP processing, thus building a foundation for future development of potential Alzheimer's disease therapeutic strategies.

Hsp90-binding immunophilin FKBP52 modulates telomerase activity by promoting the cytoplasmic retrotransport of hTERT.

Telomerase is a unique ribonucleoprotein enzyme that is required for continued cell proliferation. To generate catalytically active telomerase, human telomerase reverse transcriptase (hTERT) must translocate to the nucleus and assemble with the RNA component of telomerase. The molecular chaperones heat shock protein 90 (Hsp90) and p23 maintain hTERT in a conformation that enables nuclear translocation. However, the regulatory role of chaperones in nuclear transport of hTERT remains unclear. In this work, we demonstrate that immunophilin FK506-binding protein (FKBP)52 linked the hTERT-Hsp90 complex to the dynein-dynactin motor, thereby promoting the transport of hTERT to the nucleus along microtubules. FKBP52 interacted with the hTERT-Hsp90 complex through binding of the tetratricopeptide repeat domain to Hsp90 and binding of the dynamitin (Dyt) component of the dynein-associated dynactin complex to the peptidyl prolyl isomerase domain. The depletion of FKBP52 inhibited nuclear transport of hTERT, resulting in cytoplasmic accumulation. Cytoplasmic hTERT was rapidly degraded through ubiquitin (Ub)-dependent proteolysis, thereby abrogating telomerase activity. In addition, overexpression of dynamitin, which is known to dissociate the dynein-dynactin motor from its cargoes, reduced telomerase activity. Collectively, these results provide a molecular mechanism by which FKBP52 modulates telomerase activity by promoting dynein-dynactin-dependent nuclear import of hTERT.

Mitochondrial bioenergetics stimulates autophagy for pathological tau clearance in tauopathy neurons.

Hyperphosphorylation and aggregation of microtubule-associated tau is a pathogenic hallmark of tauopathies and a defining feature of Alzheimer's disease (AD). Pathological tau is targeted by autophagy for clearance, but autophagy dysfunction is indicated in tauopathy. While mitochondrial bioenergetic failure has been shown to precede the development of tau pathology, it is unclear whether energy metabolism deficiency is involved in tauopathy-related autophagy defects. Here, we reveal that stimulation of anaplerotic metabolism restores defective oxidative phosphorylation (OXPHOS) in tauopathy which, strikingly, leads to enhanced autophagy and pronounced tau clearance. OXPHOS-induced autophagy is attributed to increased ATP-dependent phosphatidylethanolamine biosynthesis in mitochondria. Excitingly, early bioenergetic stimulation boosts autophagy activity and reduces tau pathology, thereby counteracting memory impairment in tauopathy mice. Taken together, our study sheds light on a pivotal role of bioenergetic dysfunction in tauopathy-linked autophagy defects and suggests a new therapeutic strategy to prevent toxic tau buildup in AD and other tauopathies.

Ahnak in the prefrontal cortex mediates behavioral correlates of stress resilience and rapid antidepressant action in mice.

The prefrontal cortex (PFC) is a key neural node mediating behavioral responses to stress and the actions of ketamine, a fast-acting antidepressant. The molecular mechanisms underlying these processes, however, are not fully understood. Our recent study revealed a pivotal role of hippocampal Ahnak as a regulator of cellular and behavioral adaptations to chronic stress. However, despite its significant expression in the PFC, the contribution of cortical Ahnak to behavioral responses to stress and antidepressants remains unknown. Here, using a mouse model for chronic social stress, we find that Ahnak expression in the PFC is significantly increased in stress-resilient mice and positively correlated with social interaction after stress exposure. Conditional deletion of Ahnak in the PFC or forebrain glutamatergic neurons facilitates stress susceptibility, suggesting that Ahnak is required for behavioral resilience. Further supporting this notion, Ahnak expression in the PFC is increased after the administration of ketamine or its metabolite (2R, 6R)-hydroxynorketamine (HNK). Moreover, Ahnak deletion in forebrain glutamatergic neurons blocks the restorative behavioral effects of ketamine or HNK in stress-susceptible mice. This forebrain excitatory neuron-specific Ahnak deletion reduces the frequency of mini excitatory postsynaptic currents in layer II/III pyramidal neurons, suggesting that Ahnak may induce its behavioral effects via modulation of glutamatergic transmission in the PFC. Altogether, these data suggest that Ahnak in glutamatergic PFC neurons may be critical for behavioral resilience and antidepressant actions of ketamine or HNK in chronic social stress-exposed mice.

Identification of the HIV-1 packaging RNA sequence (Ψ) as a major determinant for the translation inhibition conferred by the HIV-1 5' UTR.

The HIV-1 5' untranslated region (UTR) contains conserved sequences and unique structural motifs associated with many steps in virus replication. Because unspliced HIV mRNA containing the full-length UTR serves as a template for replication and transcription as well as packaging genomic RNA into virion, it has been postulated that the UTR may play a role in translational regulation. However, the effect and the region(s) responsible for translation control remain controversial. We used deletion mutations of the 5' UTR region in both cell-based and in vitro assays to determine if secondary structural elements within the 5' UTR confer translation inhibition, and to identify which of these elements are involved. The results indicate clearly that the entire HIV-1 5' UTR confers translation inhibition in vitro and in cells; the Psi (Ψ) region specifically has the most translation inhibitory activity among the highly-structured elements in the HIV-1 5' UTR. Moreover, it was found that the SL4 structure in the Psi (Ψ) region is the major determinant of translation inhibition, and that elimination of the SL4 RNA sequence led to increased translation. The results suggest a functional role for the Psi element and the SL4 structure in the translational control of HIV-1 full-length mRNA.

Broad activation of the PRKN pathway triggers synaptic failure by disrupting synaptic mitochondrial supply in early tauopathy.

Mitochondrial defects are a hallmark of Alzheimer disease (AD), with pathologically phosphorylated MAPT/tau (phospho-MAPT/tau) reported to induce mitochondrial damage. Mitophagy constitutes a key pathway of mitochondrial quality control by which damaged mitochondria are sequestered within autophagosomes for lysosomal degradation. However, the mechanistic understanding of mitophagy and its association with pathologies under tauopathy conditions remains very limited. Here, we reveal that mitochondrial stress under phospho-MAPT/tau-mediated challenges broadly activates PRKN-mediated mitophagy which induces an unexpected effect - depletion of mitochondria from synaptic terminals, a characteristic feature in early tauopathy. PRKN activation accelerates RHOT1 turnover and consequently halts RHOT1-mediated mitochondrial anterograde movement, which disrupts mitochondrial supply to tauopathy synapses and thereby impairs synaptic function. Strikingly, increasing RHOT1 levels prevents synapse loss and reverses cognitive impairment in tauopathy mice by restoring synaptic mitochondrial populations. Thus, our study uncovers an important early mechanism underlying tauopathy-linked synaptic failure and opens a new avenue for specifically targeting early synaptic dysfunction in tauopathies, including AD.Abbreviations: AAV: adeno-associated virus; AD: Alzheimer disease; FTD: Frontotemporal dementia; LTP: long-term potentiation; Δψm: mitochondrial membrane potential; Phospho-MAPT/tau: hyperphosphorylated Microtubule Associated Protein Tau/tau; RHOT1: ras homolog family member T1; RNAi: RNA interference; Tg: transgenic.

The role of mitophagy in the regulation of mitochondrial energetic status in neurons.

Mitochondria are the main cellular energy powerhouses and supply most of the energy in the form of ATP to fuel essential neuronal functions through oxidative phosphorylation (OXPHOS). In Alzheimer disease (AD), metabolic and mitochondrial disruptions are an early feature preceding any histopathological and clinical manifestations. Mitochondrial malfunction is also linked to synaptic defects in early AD. Mitophagy serves as a key cellular quality control mechanism involving sequestration of damaged mitochondria within autophagosomes and their subsequent degradation in lysosomes. However, it remains largely unknown whether mitophagy is involved in the regulation of energy metabolism in neurons, and if so, whether metabolic deficiency in AD is attributed to mitophagy dysfunction. Here we reveal that mitophagy is broadly activated in metabolically enhanced neurons upon OXPHOS stimulation, which sustains high energetic activity by increasing mitochondrial turnover and hence facilitating mitochondrial maintenance. Unexpectedly, in AD-related mutant HsAPP Tg mouse brains, early stimulation of OXPHOS activity fails to correct energy deficits but exacerbates synapse loss as a consequence of mitophagy failure. Excitingly, lysosomal enhancement in AD neurons restores impaired metabolic function by promoting elimination of damaged mitochondria, protecting against synaptic damage in AD mouse brains. Taken together, we propose a new mechanism by which mitophagy controls bioenergetic status in neurons, furthering our understanding of the direct impact of mitophagy defects on AD-linked metabolic deficits and shedding light on the development of novel therapeutic strategies to treat AD by the early stimulation of mitochondrial metabolism combined with elevation of lysosomal proteolytic activity.Abbreviations: AD: Alzheimer disease; Aß: amyloid-ß; APP: amyloid beta precursor protein; AV: autophagic vacuole; CHX: cycloheximide; CYCS: cytochrome c, somatic; DIV: days in vitro; FRET: Förster resonance energy transfer; Gln, glutamine; LAMP1: lysosomal associated membrane protein 1; LE: late endosome; Mito: mitochondria; Δψm: mitochondrial membrane potential; OCR: oxygen consumption rate; OXPHOS: oxidative phosphorylation; SQSTM1/p62: sequestosome 1; RHEB: Ras homolog, mTORC1 binding; ROS: reactive oxygen species; STX1: syntaxin 1; SYP: synaptophysin; Tg: transgenic; TMRE: tetramethylrhodamine ethyl ester; TEM: transmission electron microscopy; WT: wild type.

Mitophagy in Alzheimer's Disease and Other Age-Related Neurodegenerative Diseases.

Mitochondrial dysfunction is a central aspect of aging and neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease. Mitochondria are the main cellular energy powerhouses, supplying most of ATP by oxidative phosphorylation, which is required to fuel essential neuronal functions. Efficient removal of aged and dysfunctional mitochondria through mitophagy, a cargo-selective autophagy, is crucial for mitochondrial maintenance and neuronal health. Mechanistic studies into mitophagy have highlighted an integrated and elaborate cellular network that can regulate mitochondrial turnover. In this review, we provide an updated overview of the recent discoveries and advancements on the mitophagy pathways and discuss the molecular mechanisms underlying mitophagy defects in Alzheimer's disease and other age-related neurodegenerative diseases, as well as the therapeutic potential of mitophagy-enhancing strategies to combat these disorders.

Mitophagy coordination with retrograde transport ensures the integrity of synaptic mitochondria.

Mitochondria sustain various essential functions at synaptic terminals. Synaptic mitochondria deficits have been implicated in early Alzheimer disease (AD) pathophysiology. Mitophagy, a selective autophagy for removal of damaged mitochondria, plays a key role in mitochondrial quality control in neurons. However, fundamental questions remain unanswered as to whether mitophagy regulates synaptic mitochondrial integrity and whether AD-associated early deficits in synaptic mitochondria are attributed to mitophagy failure. We have recently revealed that the integrity of synaptic mitochondria is maintained by a coordination of RHEB-mediated mitophagy with dynein- and SNAPIN-driven retrograde transport. We demonstrate that increased mitophagy initiation, coupled with defective retrograde transport, triggers mitophagy stress at AD synapses. Excitingly, SNAPIN-enhanced retrograde transport reduces synaptic mitophagy stress and ameliorates mitochondrial deficits, thereby counteracting synaptic damage in AD mouse brains. Therefore, our study provides new mechanistic insights into how mitophagy facilitates synaptic mitochondrial maintenance and how mitophagy failure exacerbates AD-linked mitochondrial defects and synaptic degeneration. Abbreviation: AD: Alzheimer disease; Aß: amyloid-ß; APP: amyloid beta precursor protein; CCCP: carbonyl cyanide m-chlorophenylhydrazone; LE: late endosome; Δψm, mitochondrial membrane potential; RHEB: Ras homolog enriched in brain; RNAi: RNA interference; shRNA: small hairpin RNA; Tg: transgenic.

NEDD8 ultimate buster-1 regulates the abundance of TRF1 at telomeres by promoting its proteasomal degradation.

The human telomeric protein TRF1 negatively regulates telomere length by inhibiting the access of telomerase to telomeres. Here, we describe a novel function of NEDD8 ultimate buster-1 (NUB1) for regulating the levels of TRF1 at telomeres. NUB1 is a NEDD8-interacting protein, which down-regulates the NEDD8 conjugation system. We showed that NUB1 physically interacts with TRF1 and promotes its degradation by the proteasome in the absence of NEDD8 conjugation. We also demonstrated that TRF1 is conjugated to NEDD8, and that neddylated TRF1 is targeted to the proteasome for degradation in a NUB1-dependent manner. These data suggest that NUB1 participates in telomere maintenance by regulating the levels of TRF1 at telomeres through both NEDD8-dependent and NEDD8-independent pathways.

PIAS1-mediated sumoylation promotes STUbL-dependent proteasomal degradation of the human telomeric protein TRF2.

The human telomeric protein TRF2 protects chromosome ends by facilitating their organization into the protective capping structure. Here we show that the stability of TRF2 is regulated via modification by the small ubiquitin-like modifiers (SUMO). TRF2 specifically interacts with and is sumoylated by PIAS1 in mammalian cells. The proteasome inhibitor stabilizes SUMO-conjugated TRF2 without affecting the level of unmodified TRF2, suggesting that SUMO conjugation is required for proteasomal degradation of TRF2. We also show that RNF4, a mammalian SUMO-targeted ubiquitin ligase, interacts with TRF2 in a SUMO-dependent manner and preferentially targets SUMO-conjugated TRF2 for ubiquitination. Collectively, our data demonstrate that the PIAS1-mediated sumoylation status of TRF2 serves as a molecular switch that controls the level of TRF2 at telomeres.

Examination of specific binding activity of aptamer RNAs to the HIV-NC by using a cell-based in vivo assay for protein-RNA interaction.

The nucleocapsid (NC) protein of the Human Immunodeficiency Virus-1 plays a key role in viral genomic packaging by specifically recognizing the Psi(Psi) RNA sequence within the HIV-1 genome RNA. Recently, a novel cell-based assay was developed to probe the specific interactions in vivo between the NC and Psi-RNA using E.coli cells (J. Virol. 81: 6151-55, 2007). In order to examine the extendibility of this cell-based assay to RNAs other than Psi-RNA, this study tested the RNA aptamers isolated in vitro using the SELEX method, but whose specific binding ability to NC in a living cellular environment has not been established. The results demonstrate for the first time that each of those aptamer RNAs can bind specifically to NC in a NC zinc finger motif dependent manner within the cell. This confirms that the cell-based assay developed for NC-Psi interaction can be further extended and applied to NC-binding RNAs other than Psi-RNA.