Aims

Neurodegeneration in Alzheimer’s disease (AD) selectively affects vulnerable neurons first. Here we sought to identify vulnerable neurons and explore the intrinsic characteristics and pTau and β-amyloid pathology underlying their vulnerability, with the long-term objective of developing neuronal subtype-specific neuroprotective therapies.

Methods

We developed a panel of 31 antibodies for imaging mass cytometry (IMC) and analysed 43 post-mortem human middle temporal gyri from healthy (Ctrl) and AD (Alz) donors carrying either the common (CV) or AD high-risk TREM2 variants (TREM2var). Images were processed and analysed with SIMPLI. Dirichlet model and Kruskal-Wallis plus Wilcoxon tests or ANOVA plus Tukey tests were used for paired group statistical analyses with p values corrected for multiple testing. A snRNAseq dataset from the same sample cohort was used to perform differential gene expression analysis of vulnerable neurons at the transcriptomic level.

Results

We found that intraneuronal β-amyloid signal (intraAβ) was higher in CtrlCV (13.76±5.95% intraAβ+ cells) and significantly decreased in AlzTREM2var (6.4±2.82%; p=0.0035). IntraAβ accumulated mostly in L3-6 GAD1+ and L5-6 RORB+ neuronal subtypes, which, compared to CtrlCV, were selectively reduced in AlzCV (-35.2% RORB+, p=0.0002) and AlzTREM2var (-58.5% GAD1+, p=0.045; -55.5% RORB+, p=0.0000). Conversely, pTau accumulation was found mostly in L3 RORB+ neurons, which increased in AlzCV and AlzTREM2var compared to controls (+23.9%, p=0.02; +44.5%, p=0.02). From the snRNAseq dataset, the neuronal cluster Exc-L5-RORB-LINC01202 matched L5-6 RORB+ intraAβ+ vulnerable neurons, based on markers expression. Compared to other snRNAseq clusters, Exc-L5-RORB-LINC01202 showed majority of autophagy-related differentially expressed genes.

Conclusions

Our results indicate RORB+ and GAD1+ neurons are vulnerable in AD and that pathological intraAβ accumulation, rather than pTau, may be initiating early neurodegeneration. We propose intraAβ is potentially due to a defective autophagy-lysosomal pathway, which we will further explore.

Aims

The retromer complex, comprising VPS35, VPS26, and VPS29, is highly conserved across evolution and retrieves cargoes within the endolysosomal pathway. Mutations in VPS35 are associated with Parkinson’s disease (PD). Early synaptic and lysosomal dysfunction accompany PD pathogenesis. Our prior research highlighted Vps29's influence on synaptic and lysosomal function in aging Drosophila brains. However, the underlying molecular mechanisms and their connections to PD remain unclear.

Methods

We conducted quantitative proteomics on Vps29 mutant flies and age-matched controls. A proximity labeling technique based on ascorbate peroxidase was employed to systematically assess retromer function in mouse primary neuron culture. To assess the translational relevance of our findings in Drosophila, we leveraged data from a large clinical-pathological human cohort.

Results

Mass spectrometry of Vps29 mutant flies revealed a substantial reduction in (i) subunits of the vacuolar ATPase, and (ii) regulators of clathrin-dependent endocytosis (e.g., Eps-15, Dap160, EndoA). These components are essential for lysosomal acidification and synaptic vesicle recycling. In aged Vps29 mutant fly brains, impaired lysosomal acidification was observed using a pH-sensitive reporter. Consistent with this, knockdown of retromer subunits in mammalian neurons triggered a loss of vacuolar ATPase subunits. Proximity labeling identified more than 300 proteins closely associated with VPS29 in mammalian neurons, including endogenous α-Synuclein and numerous other synaptic proteins. Notably our VPS29 spatial map strongly overlap with a previously published α-Synuclein interactome. Drosophila proteomics confirmed shared molecular signatures following either retromer dysfunction or α-Synuclein-induced neurodegeneration. Lastly, human postmortem brain proteomics similarly highlights coexpression of VPS29 with many regulators of energy-coupled proton transmembrane transport and synaptic transmission.

Conclusions

Our cross-species strategy establishes a highly conserved role for the retromer subunit VPS29 in synaptic transmission and lysosomal homeostasis and reveals unexpected connections with α-Synuclein mechanisms in PD.

Aims

Tau pathology is a common neuropathological feature in several neurodegenerative diseases including Alzheimer’s disease (AD). Mis-localization and hyperphosphorylation of tau lead to formation of intracellular neurofibrillary tangles (NFTs). Autophagy is a self-digesting system that maintains cellular homeostasis by degrading damaged organelles and toxic protein aggregates. In AD brains, a massive accumulation of autophagic vacuoles in neuron occurs, indicating dysregulated autophagy. To unambiguously elucidate in vivo the role of autophagy in pathogenic tau degradation, we established for the first-time excitatory neuron-specific autophagy-deficient tauopathy mice and analyzed tau pathology development in this mouse model brain.

Methods

Autophagy-deficient tauopathy mice were generated by crossing Atg7cKO mice and P301L human tau Tg4510 and T2 transgenic mice. Tau pathology was evaluated at 6- and 9-months-of-age by immunofluorescence (IF) staining and western blotting of sarkosyl insoluble fraction. To assess tau seeding property, we treated Tau RD P301S FRET Biosensor cells with brain homogenate from the mouse model. A series of behavior tests including modified Y-maze and open field have been performed.

Results

IF staining against phospho-tau revealed an increased number of NFT-like staining in autophagy-deficient tauopathy mouse brains compared to autophagy-competent tauopathy mouse brains. An increased accumulation of phospho-tau was also detected in sarkosyl insoluble fraction from autophagy-deficient tauopathy mouse brains. The tau seeding property was increased in brains from autophagy-deficient tauopathy mice. Modified Y-maze revealed a spatial memory impairment in autophagy-deficient tauopathy mice.

Conclusions

Autophagy-deficiency enhanced the seeding capacity of tau and increased tau pathology especially aggregated phosphorylated tau in the brain parenchyma. The memory impairment of tauopathy mouse model was aggravated by autophagy-deficiency. These data strongly suggest that autophagy has an important protective role in the development and spreading of tau pathology.

Aims

Alzheimer’s (AD) and Parkinson’s disease (PD) feature progressive neurodegeneration in a remarkably regionally selective manner. Post mortem studies have posited a role for cell autonomous mechanisms driving this, so we aimed to examine a live human induced pluripotent stem cell (iPSC)-derived neuronal model to see whether it can replicate the phenomenon of selective neuronal vulnerability, so to better determine disease mechanisms and therapeutic targets.

Methods

iPSC-derived neurons offer a rare opportunity to examine cell autonomous vulnerability in live human cells. iPSCs from patients with AD-related presenilin-1 mutations (n=6), PD-related leucine rich repeat kinase 2 mutations (n=6), and isogenic corrected (n=4) and healthy controls (n=4) have been differentiated into both cortical and midbrain dopaminergic neurons to enable comparison of pre-formed fibril (PFF) induced pathology in different neuronal subtypes from the same patient. We then examined lysosomal number, morphology, degradation, pH, and calcium using live imaging assays, alongside mitochondrial biology, and electrophysiology to understand underlying drivers of vulnerability in the cell types.

Results

Upon insult with alpha-synuclein PFFs, AD and PD dopaminergic neurons produce substantial Lewy-like pathology, whereas cortical neurons remain relatively resilient to alpha-synuclein aggregation, suggesting cell-type vulnerability. PSEN1-Intron 4 Deletion cortical neurons, however, had significantly elevated pathology. These lines displayed hyperactivity on microelectrode arrays and abnormal lysosomal biology, including increased LAMP1. PFF-insulted AD cortical neurons also have impaired neurite outgrowth, while PD cortical neurons are resilient.

Conclusions

These preliminary results show relative vulnerability of AD against PD cortical neurons, and dopaminergic against cortical neurons to alpha-synuclein aggregates for the first time. These suggest the selective vulnerability to proteinopathy exhibited in these diseases is reflected by the iPSC neuronal model and support the notion that cell intrinsic factors like autophagy drive vulnerability.

Aims

Phospholipase D3 (PLD3) is a lysosomal exonuclease that mainly targets mitochondrial DNA for degradation. Loss-of-function triggered by late onset Alzheimer’s disease (LOAD)-linked variants causes an autophagic/lysosomal catabolic bottleneck [1]. Spatial transcriptomic data now reveals a higher PLD3 expression in specific brain areas including the hypothalamus and hippocampus. How altered PLD3 expression herein affects these brain circuits is currently unknown.

Methods

We generated a PLD3 knockout mouse using RNP-CRISPR/Cas9 technology and crossed PLD3 knockout founder mice with APP^N-L-GF knock-in mice. Histological analyses were performed at 3, 6 and 9 months, whereas 6 months was chosen for behavior assessments and functional electrophysiological readouts. In vivo data were further supported by parallel investigations in primary hippocampal cultures derived from these models.

Results

Loss of PLD3 in APP^N-L-GF knock-in mice resulted in an altered amyloid plaque pathology from 6 months onwards, with fewer plaques but of larger size. Six-month-old mice further showed significant circadian problems and some light memory deficits as shown in a Barne’s Maze. We also replicated the observed lysosomal and autophagic pathology [1] in primary neurons, and now started elucidating to what extend organelle dyshomeostasis is linked to neuronal connectivity and synaptic transmission. First data using MEA-recordings of CA3 long-term potentiation showed that PLD3 deficiency significantly lowered the amplitude. The underlying mechanisms are currently further investigated focusing on ultrastructural alterations in synapses and correlating this with functional defects in lysosomal homeostasis.

Conclusions

We functionally connect defects in lysosomal homeostasis, originating from altered PLD3 catabolic activity, to changes in PLD3-expressing hypothalamic and hippocampal circuit functions that can potentially be linked to important clinical AD manifestations such as sleep and memory problems.

Ref. 1. Van Acker, Nat Commun. (2023) doi: 10.1038/s41467-023-38501-w.

Aims

Impairment of autophagy has been implicated in the pathogenesis of major human neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Previous studies primarily focused on disruptions of multiple stages of autophagy-lysosome pathway in affected neurons in AD and PD. However, whether and how deregulated autophagy in microglia contributes to AD and PD progression remains poorly understood. Our goal is to elucidate the role for microglial autophagy in regulating alpha-synuclein and amyloid plague levels and neuroprotection.

Methods

We applied genetic mouse models, primary microglial cultures, molecular and cell biology, imaging, proteomics, and transcriptomics approaches in our studies.

Results

We find that microglial autophagy engulfs and degrades neuron-released alpha-synuclein through TLR4 receptor signaling. Microglial autophagy protects dopaminergic neurons by the clearance of overflowing alpha-synuclein. Furthermore, we observe that autophagy is activated in microglia, particularly of disease-associated microglia (DAM) surrounding amyloid plaques in AD mouse models. Inhibition of microglial autophagy causes disengagement of microglia from amyloid plaques, suppression of DAM, and aggravation of neuropathology in AD mice. Mechanistically, autophagy restricts microglial senescence by suppressing Cdkn1a/p21^Cip1 expression and senescence-associated secretory phenotype (SASP).

Conclusions

Our study demonstrates a neuroprotective function of microglial autophagy in regulating the homeostasis of alpha-synuclein and amyloid plaques and preventing senescence; removal of senescent microglia is a promising therapeutic strategy.

Aims

Lewy bodies are neuropathologically associated with Lewy body dementia (LBD), but little is known about why they form or their role in the disease process. We previously noted Lewy bodies are a common finding in mitochondrial disease, so the present study sought to investigate whether deficient mitophagy in the context of mitochondrial dysfunction may underlie Lewy body formation.

Methods

Post-mortem tissue was obtained from the cingulate gyrus and dorsal motor nucleus of the vagal nerve (DMV) of mitochondrial disease cases with Lewy bodies, primary mitochondrial disease cases without Lewy bodies, and control cases, in addition to LBD cases as comparison. An array of mitophagy and autophagy markers were quantified in individual neurons of the cingulate gyrus and DMV using immunofluorescent analysis. Lewy bodies were also isolated from the cingulate gyrus for proteomic analysis of their constitutent proteins.

Results

No signficant differences were observed on the group level but Lewy bodies demonstrated a striking enrichment of markers of autophagic vesicles and mitochondria. Evaluation of diffuse alpha-synuclein aggregates thought to precede Lewy body formation demonstrated they were enriched only for autophagic mitochondria. Proteomic analysis of Lewy bodies demonstrated enrichment of markers of aggresomes, and this was confirmed histologically.

Conclusions

The presence of motor proteins associated with aggresomal trafficking through the microtubule network within Lewy bodies suggests that Lewy body formation is a deliberate attempt to encapsulate cellular waste within an insoluble structure, thus protecting the cell from the harmful effects of accumulated waste. One could speculate that Lewy bodies are a mechanism for spatial protein quality control, in the context of deficient autophagy.