The accumulation of pathological four-repeat (4R) tau is central to several frontotemporal dementia (FTD) subtypes, but human neuronal models amenable to high-throughput screening of 4R tau-targeting therapies remain very limited. To address this, we developed induced pluripotent stem cell (iPSC)-derived i3Neuron (i3N) lines expressing >75% 4R tau, driven by FTD splice-shifting mutations (Ser305Asn; S305N or S305N/IVS10 + 3). These neurons develop hyperphosphorylated tau and demonstrate somatodendritic mislocalization. These i3N neurons develop endogenous seed-competent tau and present pentameric formyl thiophene acetic acid-(pFTAA)-positive tau assemblies after 28 days in culture. For scalable screening, we CRISPR-engineered an HiBiT luminescence tag at the endogenous MAPT locus into the S305N/IVS10 + 3 iPSC line, enabling precise quantification of tau levels and pharmacological responses. The model responded predictably to compounds affecting tau clearance, demonstrating its suitability for drug discovery. Overall, this i3N platform recapitulates key features of 4R tauopathy and provides a robust system to identify therapeutic modulators of pathological tau.

Proteinopathies are frequently accompanied by co-pathologies, which complicate understanding the pathophysiological mechanisms of neurodegenerative diseases. In this study, we employed a well-characterised baboon cohort to investigate the occurrence of tau pathology following intrastriatal injections of two distinct α-synuclein extracts (small aggregates: SA and large aggregates: LA) derived from the brains of Parkinson's disease (PD) patients. Biochemical and histopathological analyses (6, 12, or 24 months after injections) revealed significantly higher phospho-tau expression and greater frequency of neuropil threads in both LA- (n=18) and SA-injected baboons (n=12) compared with control animals. We also observed distinct patterns of tau pathology depending on the assembly of α-synuclein aggregates. Baboons injected with SA exhibited tangle-like phosphorylated tau lesions and neuropil threads, with no significant differences across post-injection time points. Moreover, a uniform distribution of tau pathology was detected along the rostro-caudal axis of the medial temporal lobe, affecting neuronal, oligodendroglial, and astrocytic cell types. On the other hand, baboons injected with LA exhibited neuropil threads and few glial lesions but lacked phospho-tau intraneuronal aggregates. The occurrence of phospho-tau lesions was associated with phospho-synuclein pathology. These observations suggest a link between nigrostriatal synucleinopathy and limbic tau co-pathology, providing pathophysiological insights into PD dementia.

Age is the greatest risk factor for Alzheimer's disease (AD) and aging is associated with decline in multiple pathways including cellular proteostasis efficiency, which has been shown to precipitate the accumulation of pathological proteins including tau. We are interested in whether there is an intersection between neuronal populations that are selectively vulnerable to age-related proteostasis deficits and those that are vulnerable to tauopathy. These data may help explain the phenomenon of primary age-related tauopathy (PART) (doi:10.1007/s00401-014-1349-0) and provide a framework to study the relationship between PART and AD. To test this, we have used a range of spatial biology and computational techniques, including spatial cell-typing and multi-plex imaging in aged mice, and compared vulnerable neuron populations to those affected by tauopathy in new, targeted mouse models of tauopathy ( https://doi.org/10.1038/s41593-024-01829-7 ). During healthy aging, we have demonstrated that supragranular neurons from the entorhinal cortex uniquely accumulate the autophagy adaptor protein p62. Using a new spatial imaging technique, coppaFISH-3D, we identified that Layer2/3 intra-telencephalic projecting neurons of the entorhinal cortex (L2/3 IT ENT) are more likely to accumulate p62 than other L2/3 IT neurons. Those neurons are thought to be amongst the earliest ones affected by tau pathology. Building on a single-nuclei multiome dataset we showed that a gene network including synapse and calcium related genes was enriched in L2/3 IT ENT to be profoundly downregulated with aging, suggesting a functional impairment of those neurons. Such downregulation was not seen in neighbouring L2/3 IT neurons. ‘Protected’ neurons had higher expression of a proteostasis-related module, containing chaperone, lysosomal, and proteasome genes was observed. L2/3 IT ENT neurons fail to boost a proteostasis response potentially explaining p62 accumulation during ageing. We then assessed vulnerability to tau accumulation (including those phosphorylated at different epitopes) in the entorhinal cortex of a new MAPT KI mouse in situ , classifying affected neurons into fine transcriptomic subtypes. Affected neurons in the EC mapped to L2/3 IT ENT neurons. These data strongly support the idea that neuronal populations that are vulnerable to declining proteostasis in older age are also vulnerable to accumulating tau.

Chaperone-mediated autophagy (CMA) declines in ageing and neurodegenerative diseases. Loss of CMA in neurons leads to neurodegeneration and behavioural changes in mice but the role of CMA in neuronal physiology is largely unknown. Here we show that CMA deficiency causes neuronal hyperactivity, increased seizure susceptibility and disrupted calcium homeostasis. Pre-synaptic neurotransmitter release and NMDA receptor-mediated transmission were enhanced in CMA-deficient females, whereas males exhibited elevated post-synaptic AMPA-receptor activity. Comparative quantitative proteomics revealed sexual dimorphism in the synaptic proteins degraded by CMA, with preferential remodelling of the pre-synaptic proteome in females and the post-synaptic proteome in males. We demonstrate that genetic or pharmacological CMA activation in old mice and an Alzheimer's disease mouse model restores synaptic protein levels, reduces neuronal hyperexcitability and seizure susceptibility, and normalizes neurotransmission. Our findings unveil a role for CMA in regulating neuronal excitability and highlight this pathway as a potential target for mitigating age-related neuronal decline.

Tau pathology is a hallmark of several neurodegenerative diseases, including frontotemporal dementia and Alzheimer’s disease. However, the sequence of events and the form of tau that confers toxicity are still unclear, due in large part to the lack of physiological models of tauopathy initiation and progression in which to test hypotheses. We have developed a series of targeted mice expressing frontotemporal-dementia-causing mutations in the humanized MAPT gene to investigate the earliest stages of tauopathy. MAPTInt10+3G>A and MAPTS305N;Int10+3G>A lines show abundant hyperphosphorylated tau in the hippocampus and entorhinal cortex, but they do not develop seed-competent fibrillar structures. Accumulation of hyperphosphorylated tau was accompanied by neurite degeneration, loss of viable synapses and indicators of behavioral abnormalities. Our results demonstrate that neuronal toxicity can occur in the absence of fibrillar, higher-order structures and that tau hyperphosphorylation is probably involved in the earliest etiological events in tauopathies showing isoform ratio imbalance. Mice expressing humanized mutant tau exhibit synaptic loss and behavioral abnormalities in the absence of abnormal tau conformers, suggesting that hyperphosphorylated tau can be pathological, at least in tauopathies caused by tau isoform imbalance.

Lipophagy is a ubiquitous mechanism for degradation of lipid droplets (LDs) in lysosomes. Autophagy receptors selectively target organelles for lysosomal degradation. The selective receptor for lipophagy remains elusive. Using mouse liver phosphoproteomics and human liver transcriptomics, we identify vacuolar-protein-sorting-associated protein 4A (VPS4A), a member of a large family AAA+ ATPases, as a selective receptor for lipophagy. We show that phosphorylation of VPS4A on Ser95,97 and its localization to LDs in response to fasting drives lipophagy. Imaging/three-dimensional (3D) reconstruction and biochemical analyses reveal the concomitant degradation of VPS4A and LDs in lysosomes in an autophagy-gene-7-sensitive manner. Either silencing VPS4A or targeting VPS4AS95,S97 phosphorylation or VPS4A binding to LDs or LC3 blocks lipophagy without affecting other forms of selective autophagy. Finally, VPS4A levels and markers of lipophagy are markedly reduced in human steatotic livers-revealing a fundamental role of VPS4A as the lipophagy receptor in mice and humans.

Reduction of amyloid beta (Aβ) has been shown to be effective in treating Alzheimer’s disease (AD), but the underlying assumption that neurons are the main source of pathogenic Aβ is untested. Here, we challenge this prevailing belief by demonstrating that oligodendrocytes are an important source of Aβ in the human brain and play a key role in promoting abnormal neuronal hyperactivity in an AD knock-in mouse model. We show that selectively suppressing oligodendrocyte Aβ production improves AD brain pathology and restores neuronal function in the mouse model in vivo. Our findings suggest that targeting oligodendrocyte Aβ production could be a promising therapeutic strategy for treating AD.

BACKGROUND:Many putative causes and risk factors have been associated with outcomes in Alzheimer's disease (AD) but all attempts at disease-modifying treatment have failed to be clinically significant. Efforts to address this "association-intervention" mismatch have tended to focus on the novel design of interventions.OBJECTIVE:Here, we instead deal with the notion of association in depth. We introduce the concept of disease-associated process (DAP) as a flexible concept that can unite different areas of study of AD from genetics to epidemiology to identify disease-modifying targets.METHODS:We sort DAPs using three properties: specificity for AD, frequency in patients, and pathogenic intensity for dementia before using a literature review to apply these properties in three ways. Firstly, we describe and visualize known DAPs. Secondly, we exemplify qualitative specificity analysis with the DAPs of tau protein pathology and autophagy to reveal their differential implication in AD. Finally, we use DAP properties to define the terms "risk factor," "cause," and "biomarker."RESULTS:We show how DAPs fit into our collaborative disease ontology, the Alzheimer's Disease-Associated Processes and Targets (ADAPT) ontology. We argue that our theoretical system can serve as a democratic research forum, offering a more biologically adequate view of dementia than reductionist models.CONCLUSION:The ADAPT ontology is a tool that could help to ground debates around priority setting using objective criteria for the identifying of targets in AD. Further efforts are needed to address issues of how biomedical research into AD is prioritized and funded.

Fasting triggers diverse physiological adaptations including increases in circulating fatty acids and mitochondrial respiration to facilitate organismal survival. The mechanisms driving mitochondrial adaptations and respiratory sufficiency during fasting remain incompletely understood. Here we show that fasting or lipid availability stimulates mTORC2 activity. Activation of mTORC2 and phosphorylation of its downstream target NDRG1 at serine 336 sustains mitochondrial fission and respiratory sufficiency. Time-lapse imaging shows that NDRG1, but not the phosphorylation-deficient NDRG1 Ser336Ala mutant, engages with mitochondria to facilitate fission in control cells, as well as in those lacking DRP1. Using proteomics, a small interfering RNA screen, and epistasis experiments, we show that mTORC2-phosphorylated NDRG1 cooperates with small GTPase CDC42 and effectors and regulators of CDC42 to orchestrate fission. Accordingly, Rictor KO , NDRG1 Ser336Ala mutants and Cdc42 -deficient cells each display mitochondrial phenotypes reminiscent of fission failure. During nutrient surplus, mTOR complexes perform anabolic functions; however, paradoxical reactivation of mTORC2 during fasting unexpectedly drives mitochondrial fission and respiration.

INTRODUCTION:The "prion-like" features of Alzheimer's disease (AD) tauopathy and its relationship with amyloid-β (Aβ) have never been experimentally studied in primates phylogenetically close to humans. METHODS:We injected 17 macaques in the entorhinal cortex with nanograms of seeding-competent tau aggregates purified from AD brains or control extracts from aged-matched healthy brains, with or without intracerebroventricular co-injections of oligomeric-Aβ. RESULTS:Pathological tau injection increased cerebrospinal fluid (CSF) p-tau181 concentration after 18 months. Tau pathology spreads from the entorhinal cortex to the hippocampal trisynaptic loop and the cingulate cortex, resuming the experimental progression of Braak stage I to IV. Many AD-related molecular networks were impacted by tau seeds injections regardless of Aβ injections in proteomic analyses. However, we found mature neurofibrillary tangles, increased CSF total-tau concentration, and pre- and postsynaptic degeneration only in Aβ co-injected macaques. DISCUSSION:Oligomeric-Aβ mediates the maturation of tau pathology and its neuronal toxicity in macaques but not its initial spreading. HIGHLIGHTS:This study supports the "prion-like" properties of misfolded tau extracted from AD brains. This study empirically validates the Braak staging in an anthropomorphic brain. This study highlights the role of oligomeric Aβ in driving the maturation and toxicity of tau pathology. This work establishes a novel animal model of early sporadic AD that is closer to the human pathology.

Aggregation of α-synuclein (α-syn) is the cornerstone of neurodegenerative diseases termed synucleinopathies, which include Parkinson’s Disease (PD), Dementia with Lewy Bodies (DLB), and Multiple System Atrophy (MSA). These synucleinopathies are characterized by the deposit of aggregated α-syn in intracellular inclusions observable in neurons and glial cells. In PD and DLB, these aggregates, predominantly located in neurons, are called Lewy Bodies (LBs). These LBs are one of the pathological hallmarks of PD and DLB, alongside dopaminergic neuron loss in the substantia nigra. Previous studies have demonstrated the ability of PD patient-derived LB fractions to induce nigrostriatal neurodegeneration and α-syn pathology when injected into the striatum or the enteric nervous system of non-human primates. Here, we report the pathological consequences of injecting these LB fractions into the cortex of non-human primates. To this end, we inoculated mesencephalic PD patient-derived LB fractions into the prefrontal cortex of baboon monkeys terminated one year later. Extensive analyses were performed to evaluate pathological markers known to be affected in LB pathologies. We first assessed the hypothesized presence of phosphorylated α-syn at S129 (pSyn) in the prefrontal cortices. Second, we quantified the neuronal, microglial, and astrocytic cell survival in the same cortices. Third, we characterized these cortical LB injections’ putative impact on the integrity of the nigrostriatal system. Overall, we observed pSyn accumulation around the injection site in the dorsal prefrontal cortex, in connected cortical regions, and further towards the striatum, suggesting α-syn pathological propagation. The pathology was also accompanied by neuronal loss in these prefrontal cortical regions and the caudate nucleus, without, however, loss of nigral dopamine neurons. In conclusion, this pilot study provides novel data demonstrating the toxicity of patient-derived extracts, their potential to propagate from the cortex to the striatum in non-human primates, and a possible primate model of DLB.

Two potential disease-modifying approaches for dementia are being vigorously tested: the early targeting of the neuropathology of Alzheimer's disease (AD) and multi-domain lifestyle interventions to promote resilience to neuropathology. We apply the "web of information" model of clinical translation to both approaches to argue firstly that tests of treatments aiming to achieve clinically meaningful outcomes should remain simple, and secondly, that building clinically-meaningful treatments should be kept separate from public health policy which means promoting wide-reaching action against risk factors now with available information.

Defective clearance of tau by neuronal autophagy has been implicated in the accumulation of toxic forms of tau in Alzheimer’s disease (AD). A novel, endogenous activator of chaperone-mediated autophagy (CMA-modulator) was identified in cell culture experiments (Cuervo laboratory, unpublished data). It is unclear what role this endogenous CMA-modulator plays in AD. We hypothesized that loss of the CMA-modulator in neurons could lead to decreased clearance of abnormal tau monomers, promoting the accumulation of toxic forms of tau. This study examined the proportion of neurons with CMA-modulator in postmortem brain tissue from subjects with a range of AD neuropathologic changes as well as both amnestic and non-amnestic AD presentation. We used multiplex immunofluorescence (CMA-modulator, NeuN for neurons, 1F3C for acetyl-tau, AT100 for phospho-tau) on tissue microarrays in the inferior temporal gyrus of 17 subjects (Table 1) and for each subject quantified neurons within a gray matter area of 7.4 - 7.6 mm 2 using digital pathology. To analyze the change in percentage of neurons positive for CMA-modulator across Braak stages, we fit a linear regression model. In amnestic AD, the percentage of neurons positive for CMA-modulator declined linearly with advancing Braak stage, but this trend was not statistically significant (slope = 2.42% loss per Braak stage, p = 0.086). The predicted percentage of positive neurons was 22.8% at Braak stage 0 and 8.25% at Braak stage 6 and amnestic presentation. All non-amnestic cases were Braak stage 6, and the proportion of neurons positive for the CMA-modulator varied greatly from 0.2% to 77.1%. There was no significant association between the percentage of CMA-modulator positive neurons and either age or gender. Quantification for the acetyl-tau and phospho-tau markers is ongoing. There may be a gradual loss of the endogenous CMA-modulator across Braak stages in amnestic AD, but this study was underpowered to draw definitive conclusions. We are increasing the sample size.

The term autophagy encompasses different pathways that route cytoplasmic material to lysosomes for degradation and includes macroautophagy, chaperone-mediated autophagy, and microautophagy. Since these pathways are crucial for degradation of aggregate-prone proteins and dysfunctional organelles such as mitochondria, they help to maintain cellular homeostasis. As post-mitotic neurons cannot dilute unwanted protein and organelle accumulation by cell division, the nervous system is particularly dependent on autophagic pathways. This dependence may be a vulnerability as people age and these processes become less effective in the brain. Here, we will review how the different autophagic pathways may protect against neurodegeneration, giving examples of both polygenic and monogenic diseases. We have considered how autophagy may have roles in normal CNS functions and the relationships between these degradative pathways and different types of programmed cell death. Finally, we will provide an overview of recently described strategies for upregulating autophagic pathways for therapeutic purposes.

Significance Cardiovascular diseases remain the leading cause of death worldwide, with atherosclerosis being the most common source of clinical events. Metabolic changes with aging associate with concurrent increased risk of both type 2 diabetes and cardiovascular disease, with the former further raising the risk of the latter. The activity of a selective type of autophagy, chaperone-mediated autophagy (CMA), decreases with age or upon dietary excesses. Here we study whether reduced CMA activity increases risk of atherosclerosis in mouse models. We have identified that CMA is up-regulated early in response to proatherogenic challenges and demonstrate that reduced systemic CMA aggravates vascular pathology in these conditions. We also provide proof-of-concept support that CMA up-regulation is an effective intervention to reduce atherosclerosis severity and progression.

Synucleinopathies encompass several neurodegenerative diseases, which include Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. These diseases are characterized by the deposit of α-synuclein aggregates in intracellular inclusions in neurons and glial cells. Unlike Parkinson's disease and dementia with Lewy bodies, where aggregates are predominantly neuronal, multiple system atrophy is associated with α-synuclein cytoplasmic inclusions in oligodendrocytes. Glial cytoplasmic inclusions are the pathological hallmark of multiple system atrophy and are associated with neuroinflammation, modest demyelination and, ultimately, neurodegeneration. To evaluate the possible pathogenic role of glial cytoplasmic inclusions, we inoculated glial cytoplasmic inclusion-containing brain fractions obtained from multiple system atrophy patients into the striatum of non-human primates. After a 2-year in vivo phase, extensive histochemical and biochemical analyses were performed on the whole brain. We found loss of both nigral dopamine neurons and striatal medium spiny neurons, as well as loss of oligodendrocytes in the same regions, which are characteristics of multiple system atrophy. Furthermore, demyelination, neuroinflammation and α-synuclein pathology were also observed. These results show that the α-synuclein species in multiple system atrophy-derived glial cytoplasmic inclusions can induce a pathological process in non-human primates, including nigrostriatal and striatofugal neurodegeneration, oligodendroglial cell loss, synucleinopathy and gliosis. The present data pave the way for using this experimental model for MSA research and therapeutic development.

Summary Fasting triggers diverse cellular and metabolic adaptations to facilitate organismal survival 1,2 . During nutrient deprivation, increases in circulating fatty acids support mitochondrial respiration 2 . The mechanisms driving mitochondrial adaptations and respiratory sufficiency during nutrient deprivation remain incompletely understood. Here we show that extended periods of fasting, or lipid availability stimulates mTORC2 activity. Activation of mTORC2 and phosphorylation of its target NDRG1 3 at S336 sustains mitochondrial fission and respiratory sufficiency. Timelapse imaging reveals that wildtype NDRG1, but not phosphorylation-deficient NDRG1 S336A mutant, engages with mitochondria to facilitate its scission. Using proteomics, and an siRNA screen, we show that mTORC2-phosphorylated NDRG1 cooperates with the small GTPase Cdc42 4 and Cdc42-specific effectors and regulators to orchestrate fission. Accordingly, Rictor KO , NDRG1 S336A mutants, and Cdc42 -deficient cells each display mitochondrial phenotypes reminiscent of fission failure. During nutrient surplus, mTOR complexes perform anabolic functions 5 ; however, paradoxical reactivation of mTORC2 during fasting plays an unexpected role in driving mitochondrial fission and respiration.