Welcome to the AD/PD™ 2021 Interactive Program
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- Live Session | 
- On Demand Session | 
- On Demand with Live Q&A
The viewing of sessions, cannot be accessed from this conference calendar. All sessions are accessible via the Main Lobby.
FOLLOWING THE LIVE DISCUSSION, THE RECORDING WILL BE AVAILABLE IN THE ON-DEMAND SECTION OF THE AUDITORIUM.
TFEB-MEDIATED LYSOSOMAL REGULATION AND SIGNALING IN ALZHEIMER’S DISEASE
Abstract
Abstract Body
Neurofibrillary tangles composed of aggregates of hyper-phosphorylated tau protein is a key pathological hallmark of Alzheimer’s disease (AD). We found that the Transcription Factor EB (TFEB), a major regulator of the autophagy-lysosomal pathway, plays a critical role in addressing tau pathology and rescue of neurodegeneration in mouse models. We demonstrate that neuronal TFEB exhibits a potent effect in the clearance of neurofibrillary tangles through autophagy and lysosomal exocytosis while astroglial TFEB plays a non-cell-autonomous role in preventing the cell-to-cell spreading of tau pathology. Analysis of gene expression profiles in both tau mouse models and human AD brains revealed an enrichment of lysosomal genes, in particular the genes controlled by TFEB, suggesting that this lysosomal response to disease is conserved in mice and humans. These findings along with our in vitro work suggest that tau pathology triggers lysosomal stress and activates TFEB as a protective response against ongoing cellular stress. Finally, by genetically manipulating TFEB’s regulation of the lysosomal proton pump (vATPase), we show a critical role of the TFEB-mediated lysosomal stress response in modulating disease progression. These findings provide a mechanistic framework of the TFEB-lysosomal network’s role in lysosomal homeostasis and AD pathogenesis.
MICROGLIAL AUTOPHAGY: A NEUROPROTECTIVE MECHANISM IN PARKINSON’S AND ALZHEIMER’S DISEASE
Abstract
Aims
Microglia maintain brain homeostasis by removing neuron-derived components such as myelin, cell debris and synapse. The evidence linking microglia to neurodegenerative diseases such as Alzheimer’s (AD) and Parkinson’s disease (PD) is growing, however, the precise mechanisms remain poorly understood. A common pathological hallmark of AD, PD and other age-related neurodegenerative diseases is the accumulation of protein aggregates as a result of disruption of proteostasis in aged brains. Autophagy, as a major cellular bulk degradation pathway, is known to protect neurons through cell autonomous clearance of protein and membrane associated cargo in neuron. Whether or not autophagy plays a significant role in neuroprotective function of microglia, however, remains elusive. Our aim is to dissect pathophysiological function of microglial autophagy in the molecular mechanism underlying Parkinson’s and Alzheimer’s disease.
Methods
We used interdisciplinary approaches including genetic animal models, primary cultures, 3D-image construction, electron microscopy, and protein biochemistry in our study.
Results
We have recently investigated physiology of microglial autophagy in CNS and uncovered the mechanism for how neuron produced alpha-synuclein and A-beta activate and mobilize microglial autophagy in protection through digestion.
Conclusions
Our study provides insight into how a conserved lysosome degradation mechanism controls disease protein aggregate formation, spreading and clearance, therefore identifying a novel therapeutic target of PD and AD.
A FUNCTIONAL ROLE FOR UBIQUILIN-2 IN REGULATING LEVELS OF ALPHA-SYNUCLEIN
Abstract
Aims
The protein quality control protein, ubiquilin-2 (UBQLN2), co-localizes with α-synuclein aggregates in Parkinson’s disease (PD) and Lewy body dementia (LBD), implicating UBQLN2 in synucleinopathies. However, little is known about how it may interact with and clear α-synuclein. This study aimed to define the role of UBQLN2 in handling α-synuclein.
Methods
To evaluate whether UBQLN2 regulates α-synuclein clearance in vitro and in vivo, we used western blot to measure α-synuclein or pS129 α-synuclein levels in HEK-293 cells transiently expressing or deleted of UBQLN2 and in multiple transgenic mouse lines including: UBLQN2 overexpressing mice (Ub2-hi) and A53T α-synuclein mice crossed with Ub2-hi or Ub2-KO mice. We measured soluble and insoluble UBQLN2 levels in human brain lysates from PD and LBD brains to assess solubility. To determine whether UBQLN2’s action on α-synuclein is ubiquitin-dependent, we measured α-synuclein levels following transient transfection of an engineered mutant UBQLN2 (L619A) that cannot bind ubiquitin.
Results
In vitro, UBQLN2 significantly decreased soluble α-synuclein levels. In vivo, α-synuclein levels decreased in UBQLN2-overexpressing mice. A53T α-synuclein and pS129 levels were unchanged in A53TxUb2-hi mice versus A53T controls but were significantly increased in A53TXUb2-KO mice. Human brain tissue studies revealed increased insoluble UBQLN2 levels in PD and LBD. Unexpectedly, L619A UBQLN2 was more effective than wild-type UBQLN2 at reducing α-synuclein levels, implying a ubiquitin-independent effect.
Conclusions
Our results support a functional role for UBQLN2 in regulating a-synuclein levels and that UBQLN2 solubility is altered in PD and LBD disease brains. Ongoing studies seek to elucidate the mechanism by which UBQLN2 helps clear a-synuclein.
MICRO-RNA ALTERATIONS DETECTABLE IN CSF EXTRACELLULAR VESICLES RELATED TO DYSFUNCTION OF AUTOPHAGY PATHWAYS IN 4R-TAUOPATHIES
Abstract
Aims
Our goals were: 1) to identify microRNA alterations in cerebrospinal fluid (CSF) extracellular vesicles (EVs) from patients with frontotemporal disorders; 2) to elucidate the effect of candidate microRNAs on autophagy mechanisms and tau turnover.
Methods
EV-derived microRNA levels from 144 CSF samples (including healthy controls, patients with Alzheimer’s disease (AD) or frontotemporal disorders) were analyzed by qPCR. MicroRNA changes in patients were reproduced in two in-vitro models. Total RNA isolated from SK-N-MC cells quantified by RNA-seq. Neuro-2a cells with inducible expression of human wild-type (WT)-tau or pathogenic P301L-tau were used to measure levels of tau and autophagy-related proteins by immunoblot. Neuro-2a cells expressing fluorescent reporters were used to monitor autophagy dynamics.
Results
MicroRNA profiling in CSF-derived EVs revealed four microRNAs differentially expressed in 4R tau-related pathologies when compared to other phenotypes. Analysis of gene expression profiles upon imposing changes in miR that mimic those found in the patients’ CSF revealed 30 genes differentially expressed for three candidate microRNAs, which point to 17 significantly enriched biological pathways. Quantification of turnover of WT and mutant tau in Neuro-2a cell lines, after treatment with microRNA modulators, suggests that autophagic degradation of tau is impaired when reproducing certain microRNA alterations observed in patients. Autophagy reporter studies also indicate that microRNA changes observed in the patients are inhibitory in some autophagy pathways.
Conclusions
Our results indicate the presence of a characteristic microRNA signature seemingly specific for 4R-tauopathies. Functional studies of these microRNAs in cultured cells showed inhibitory effects on some autophagy pathways and impairment of tau turnover.
NONCANONICAL FUNCTION OF THE AUTOPHAGY MACHINERY PREVENTS SPONTANEOUS AGE-ASSOCIATED ALZHEIMER’S DISEASE & NEUROINFLAMMATION
Abstract
Aims
Non-canonical functions of autophagy proteins have been implicated in neurodegenerative conditions, including Alzheimer’s Disease (AD). We previously identified a novel role for the autophagy machinery in regulating the conjugation of LC3 to endosomes, a process we termed LC3-associated endocytosis (LANDO). We showed abrogation of LANDO resulted in massive exacerbation of disease pathology including neuroinflammation and neurodegeneration in the 5xFAD mouse model. While these results demonstrated a role for LANDO in mitigation of disease driven by overexpression/humanization, we aimed to evaluate the role of LANDO in mitigation of AD endogenously in mice.
Methods
We identified that the WD-domain of the autophagy protein Atg16L is dispensable for canonical autophagy but required for its non-canonical functions, including regulation of LANDO. We aged LANDO-deficient, Atg16L WD-domain deficient mice to evaluate consequences of LANDO-deficiency with age.
Results
Two-year-old mice lacking this domain presented with robust Aβ-pathology, tau hyperphosphorylation, reactive microgliosis, pervasive neurodegeneration, and severe behavioral and memory deficiency, consistent with human disease. Mechanistically, we found that this WD-domain was required for the recycling of β-amyloid receptors in primary microglia. Further, pharmacologic suppression of neuroinflammation reversed established memory impairment and markers of disease pathology in this novel AD model.
Conclusions
The loss of the Atg16L WD-domain and LANDO drives spontaneous AD in mice, and inhibition of neuroinflammation is a potential therapeutic approach for treating neurodegeneration and memory loss. A decline in expression of ATG16L in the brains of human AD patients suggests the possibility that a similar mechanism may contribute to disease in human AD.
ACIDIC NANOPARTICLES PROTECT AGAINST ALPHA-SYNUCLEIN-INDUCED NEURODEGENERATION THROUGH RESTORATION OF LYSOSOMAL ACTIVITY
Abstract
Aims
Parkinson disease is a neurodegenerative disease characterized by accumulation of alpha-synuclein protein enclaved in neuronal intracytoplasmic inclusions called Lewy Bodies. Genetic and neuropathological evidences indicate an alteration of the autophagy-lysosomal pathway at different levels. A likely link between these data relates to a lysosomal impairment, making it a point of particular vulnerability. To restore the autophagy-lysosomal pathway function, we chose to target the lysosomal compartment using acidic nanoparticles aiming to reestablish the appropriate and functional lysosomal pH in a PD mouse model.
Methods
We produced acidic nanoparticles made of biocompatible poly(D,L-lactide-co-glycolide) polymers, previously shown to be efficient to target lysosomal compartment and reestablish proper lysosomal pH in vitro. To test this hypothesis, we used the Lewy body mouse model of PD which consist of mice receiving human derived Lewy body containing fractions into the substantia nigra. We then injected acidic nanoparticles or non-acidic nanoparticles in the substantia nigra of Lewy body-injected or control mice.
Results
Four months post-injection, we evaluated the extent of the nigrostriatal lesions and demonstrated an attenuation of dopaminergic neurodegeneration in these animals, both at the level of nigral dopaminergic neuron cell bodies and striatal dopaminergic terminals and we showed decreased levels of total, proteinase K resistant and S129 phosphorylated alpha synuclein in the substantia nigra of acidic nanoparticles and Lewy body injected mice. In addition, we show that acidic nanoparticles increase alpha synuclein degradation through enhancing lysosomal activity.
Conclusions
In conclusion, our results support lysosomal reacidification as a disease-modifying strategy for the treatment of Parkinson disease and other synucleinopathies.
EXTRACELLULAR MATRIX PROTEIN DECORIN IS INCREASED IN CSF OF APP KNOCK-IN MICE AND EARLY STAGE OF ALZHEIMER’S DISEASE
Abstract
Aims
Alzheimer’s disease (AD) brains are characterized by extracellular amyloid-beta (Aβ) deposition and autophagy dysregulation. Here we aimed at deepening the understanding of these brain pathologies and how they translate to the CSF.
Methods
AD postmortem brain tissues and App knock-in mouse models (AppNL-F and AppNL-G-F) were used for autophagy characterization. The cerebrospinal fluid (CSF) from App knock-in mice was analyzed by label-free mass spectrometry (MS) and compared with previously reported CSF MS results from patients. The expression of decorin in the mouse brains was analyzed by immunohistochemistry and western blot. The autophagy-activating effect of decorin was measured in the mouse primary neurons.
Results
p62 and LC3 II were increased in AppNL-G-F mice similar to the marked accumulation of p62 in AD brains. Several extracellular matrix (ECM) proteins, including decorin, were significantly increased in the CSF of both AppNL-Fmice and normal cognition a+t- human subjects. Decorin was mainly expressed in CA2 pyramidal neurons and parvalbumin-positive interneurons, the length of which was decreased in App knock-in mice. Decorin-treated mouse primary neurons exhibited lowered p62 and LC3 II levels.
Conclusions
Autophagy is similarly inhibited in the brains of AppNL-G-F mice and AD patients. Mice-human CSF alterations converge on several ECM proteins suggesting the alteration in blood brain barrier /blood CSF barrier composition. The ECM protein decorin activates neuronal autophagy by increasing autophagosomal-lysosomal degradation linking changes in ECM to autophagy.
AMYLOID-LIKE AGGREGATES CAUSE LYSOSOMAL DEFECTS IN NEURONS VIA GAIN-OF-FUNCTION TOXICITY
Abstract
Aims
Impairment of the autophagy-lysosomal pathway is a crucial common pathogenic mechanism in diseases characterized by protein aggregation and neurodegeneration. However, the link between aggregation and lysosomal dysfunction remains poorly understood. Here, we use artificial amyloid-like β-sheet proteins (β proteins) to investigate the gain-of-function effects of protein aggregation in primary neurons.
Methods
We use a combination of last-generation cryo-electron tomography (cryo-ET), state-of-the-art proteomics, and cell biology assays in Hela cells and primary mouse neurons.
Results
β proteins impair cellular morphology and cause toxicity in primary neurons. Cryo-ET and light microscopy experiments show that β protein aggregation leads to the accumulation of enlarged, cargo-rich autophagolysosomes; alterations reminiscent of lysosomal storage disorders. Moreover, biochemical assays point to an impairment at late stages of the autophagy-lysosomal pathway. Mass spectrometry-based analysis of the β protein interactome shows that β proteins sequester AP-3μ1, a subunit of the AP-3 adaptor complex involved in protein trafficking to lysosomal organelles. Other AP-3 subunits have been previously found within aggregates of natural disease-related proteins, and mutations in AP-3 subunits are known to cause defects of lysosomal organelles in human patients. Importantly, restoring AP-3μ1 expression ameliorates neurotoxicity caused by β proteins.
Conclusions
We propose that impaired trafficking of lysosomal proteins due to an insufficient cellular pool of intact AP-3 complex may contribute to lysosomal defects and neurotoxicity caused by protein misfolding. Our results emphasize the toxic gain-of-function roles of protein aggregates in lysosomal dysfunction and uncover a new molecular link between protein aggregation and lysosomal defects in neurons.