Characterising the effects of genetic risk factors of Alzheimer’s disease on synaptic transmission: a functional and structural analysis

Pathological changes in neural network activity play a key role in Alzheimer’s Disease but the underlying processes remain unclear. Key genetic risk factors are correlated with the abnormal increase in production of the A1-42 peptide and the expression of the E4 isoform of the Apolipoprotein E. We began by characterizing how elevated levels of A1- 42 affect pre-synaptic activity. Employing a functional approach coupled with a strong EM-based ultrastructure readout in CA3-CA1 hippocampal synapses derived from the AD transgenic mouse model APPSwe/Ind, we show an enhancement in the recycling fraction when compared to WT terminals. Spatial analysis of the retrieved vesicles revealed a preferential localization of the functional pool in APPSwe/Ind synapses around the peri-active zone (AZ) area, suggesting an organizational correlate of functional retrieval deficits. Complementary experiments monitoring glutamate activity using the genetically encoded reporter iGluSnFr showed that A1-42 treatment over the course of 24h was sufficient to bring about a deficit in neurotransmitter release and clearance; the reported impairments were exacerbated after long-term (96h-120h) incubation. By using pharmacological interventions aimed at dampening synaptic over- activity (Levetiracetam) and improving vesicle turn-over (Roscovitine) we were able to partially rescue the reported deficits in neurotransmitter activity. Using the sypHy reporter, 24h incubation with addition of oligomeric A1-42 revealed deficits in vesicle turn-over in ApoE-transgenic mice-derived ApoE3 cultures, while ApoE4 neurons displayed no effect, while ApoE-transgenic mice reported a propensity of ApoE4 animals to maintain an elevated level of functional vesicles at a later age (8 months). Our findings suggest that synapses show hyperactive function in activity-evoked vesicle recruitment with A1-42 treatment but that retrieval pathways become overwhelmed, significantly limiting ongoing signaling. Further investigation on the A1-42 dependent spatial and functional deficits are of vital importance to further elucidate the role of A1-42 in AD and intervene with therapeutic approaches

The involvement of dityrosine crosslinking in α-synuclein assembly and deposition in Lewy Bodies in Parkinson’s disease

Parkinson’s disease (PD) is characterized by intracellular, insoluble Lewy bodies composed of highly stable α-synuclein (α-syn) amyloid fibrils. α-synuclein is an intrinsically disordered protein that has the capacity to assemble to form β-sheet rich fibrils. Oxidiative stress and metal rich environments have been implicated in triggering assembly. Here, we have explored the composition of Lewy bodies in post-mortem tissue using electron microscopy and immunogold labeling and revealed dityrosine crosslinks in Lewy bodies in brain tissue from PD patients. In vitro, we show that dityrosine cross-links in α-syn are formed by covalent ortho-ortho coupling of two tyrosine residues under conditions of oxidative stress by fluorescence and confirmed using mass-spectrometry. A covalently cross-linked dimer isolated by SDS-PAGE and mass analysis showed that dityrosine dimer was formed via the coupling of Y39-Y39 to give a homo dimer peptide that may play a key role in formation of oligomeric and seeds for fibril formation. Atomic force microscopy analysis reveals that the covalent dityrosine contributes to the stabilization of α-syn assemblies. Thus, the presence of oxidative stress induced dityrosine could play an important role in assembly and toxicity of α-syn in PD

Tau (297‐391) forms filaments that structurally mimic the core of paired helical filaments in Alzheimer’s disease brain

The constituent paired helical filaments (PHFs) in neurofibrillary tangles are insoluble intracellular deposits central to the development of Alzheimer’s disease (AD) and other tauopathies. Full‐length tau requires the addition of anionic cofactors such as heparin to enhance assembly. We have shown that a fragment from the proteolytically stable core of the PHF, tau 297‐391 known as ‘dGAE’, spontaneously forms cross‐β‐containing PHFs and straight filaments under physiological conditions. Here, we have analysed and compared the structures of the filaments formed by dGAE in vitro with those deposited in the brains of individuals diagnosed with AD. We show that dGAE forms PHFs that share a macromolecular structure similar to those found in brain tissue. Thus, dGAEs may serve as a model system for studying core domain assembly and for screening for inhibitors of tau aggregation

Elevated amyloid beta disrupts the nanoscale organization and function of synaptic vesicle pools in hippocampal neurons

Alzheimer’s disease is linked to increased levels of amyloid beta (Aβ) in the brain, but the mechanisms underlying neuronal dysfunction and neurodegeneration remain enigmatic. Here, we investigate whether organizational characteristics of functional presynaptic vesicle pools, key determinants of information transmission in the central nervous system, are targets for elevated Aβ. Using an optical readout method in cultured hippocampal neurons, we show that acute Aβ42 treatment significantly enlarges the fraction of functional vesicles at individual terminals. We observe the same effect in a chronically elevated Aβ transgenic model (APPSw,Ind) using an ultrastructure-function approach that provides detailed information on nanoscale vesicle pool positioning. Strikingly, elevated Aβ is correlated with excessive accumulation of recycled vesicles near putative endocytic sites, which is consistent with deficits in vesicle retrieval pathways. Using the glutamate reporter, iGluSnFR, we show that there are parallel functional consequences, where ongoing information signaling capacity is constrained. Treatment with levetiracetam, an antiepileptic that dampens synaptic hyperactivity, partially rescues these transmission defects. Our findings implicate organizational and dynamic features of functional vesicle pools as targets in Aβ-driven synaptic impairment, suggesting that interventions to relieve the overloading of vesicle retrieval pathways might have promising therapeutic value

Tau‐mediated synaptic dysfunction is coupled with HCN channelopathy

INTRODUCTION: In tauopathies, altered tau processing correlates with impairments in synaptic density and function. Changes in hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels contribute to disease‐associated abnormalities in multiple neurodegenerative diseases. METHODS: To investigate the link between tau and HCN channels, we performed histological, biochemical, ultrastructural, and functional analyses of hippocampal tissues from Alzheimer's disease (AD), age‐matched controls, Tau35 mice, and/or Tau35 primary hippocampal neurons. RESULTS: Expression of specific HCN channels is elevated in post mortem AD hippocampus. Tau35 mice develop progressive abnormalities including increased phosphorylated tau, enhanced HCN channel expression, decreased dendritic branching, reduced synapse density, and vesicle clustering defects. Tau35 primary neurons show increased HCN channel expression enhanced hyperpolarization‐induced membrane voltage “sag” and changes in the frequency and kinetics of spontaneous excitatory postsynaptic currents. DISCUSSION: Our findings are consistent with a model in which pathological changes in tauopathies impact HCN channels to drive network‐wide structural and functional synaptic deficits. Highlights: - Hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels are functionally linked to the development of tauopathy. - Expression of specific HCN channels is elevated in the hippocampus in Alzheimer's disease and the Tau35 mouse model of tauopathy. - Increased expression of HCN channels in Tau35 mice is accompanied by hyperpolarization‐induced membrane voltage “sag” demonstrating a detrimental effect of tau abnormalities on HCN channel function. - Tau35 expression alters synaptic organization, causing a loosened vesicle clustering phenotype in Tau35 mice

Dityrosine cross-links are present in alzheimer's disease-derived Tau Oligomers and Paired Helical Filaments (PHF) which Promotes the stability of the PHF-core Tau (297–391) in vitro

A characteristic hallmark of Alzheimer's Disease (AD) is the pathological aggregation and deposition of tau into paired helical filaments (PHF) in neurofibrillary tangles (NFTs). Oxidative stress is an early event during AD pathogenesis and is associated with tau-mediated AD pathology. Oxidative environments can result in the formation of covalent dityrosine crosslinks that can increase protein stability and insolubility. Dityrosine cross-linking has been shown in Aβ plaques in AD and α-synuclein aggregates in Lewy bodies in ex vivo tissue sections, and this modification may increase the insolubility of these aggregates and their resistance to degradation. Using the PHF-core tau fragment (residues 297 – 391) as a model, we have previously demonstrated that dityrosine formation traps tau assemblies to reduce further elongation. However, it is unknown whether dityrosine crosslinks are found in tau deposits in vivo in AD and its relevance to disease mechanism is unclear. Here, using transmission electron microscope (TEM) double immunogold-labelling, we reveal that neurofibrillary NFTs in AD are heavily decorated with dityrosine crosslinks alongside tau. Single immunogold-labelling TEM and fluorescence spectroscopy revealed the presence of dityrosine on AD brain-derived tau oligomers and fibrils. Using the tau (297–391) PHF-core fragment as a model, we further showed that prefibrillar tau species are more amenable to dityrosine crosslinking than tau fibrils. Dityrosine formation results in heat and SDS stability of oxidised prefibrillar and fibrillar tau assemblies. This finding has implications for understanding the mechanism governing the insolubility and toxicity of tau assemblies in vivo

Paired helical filament-forming region of tau (297–391) influences endogenous tau protein and accumulates in acidic compartments in human neuronal cells

Assembly of tau protein into paired helical filaments and straight filaments is a key feature of Alzheimer's disease. Aggregation of tau has been implicated in neurodegeneration, cellular toxicity and the propagation, which accompanies disease progression. We have reported previously that a region of tau (297–391), referred to as dGAE, assembles spontaneously in physiological conditions to form paired helical filament-like fibres in vitro in the absence of additives such as heparin. This provides a valuable tool with which to explore the effects of tau in cell culture. Here we have studied the cellular uptake of soluble oligomeric and fibrillar forms of dGAE and examined the downstream consequences of tau internalisation into differentiated SH-SY5Y neuroblastoma cells using fluorescence and electron microscopy alongside structural and biochemical analyses. The assembled dGAE shows more acute cytotoxicity than the soluble, non-aggregated form. Conversely, the soluble form is much more readily internalised and, once within the cell, is able to associate with endogenous tau resulting in increased phosphorylation and aggregation of endogenous tau, which accumulates in lysosomal/endosomal compartments. It appears that soluble oligomeric forms are able to propagate tau pathology without being acutely toxic. The model system we have developed now permits the molecular mechanisms of propagation of tau pathology to be studied in vitro in a more physiological manner with a view to development of novel therapeutic approaches

Research data for paper: Elevated amyloid beta disrupts the nanoscale organization and function of synaptic vesicle pools in hippocampal neurons

Data for paper published in Cerebral Cortex on 03/04/22 Files: Biasettietal-VPools-TEM (Transmission electron microscope)-maps: TEM-based vesicle pool maps by synapse and condition, used to generate distance plots. Biasettietal-SypHy-fractions: Fluorescence SypHy values used to calculate pool fractions by synapse and condition. Biasettietal-iGluSNFR: iGluSnFR response values by synapse, experiment and condition. Biasettietal-EMdistVtoAZ: Coordinates of each vesicle from TEM images measured as distances to nearest point on the active zone; this is used to generate mean distances for each pool class (PC+: photoconverted, PC-: non-photoconverted) and also to construct cumulative distance plots; measurements were collected for both WT (wild-type) and APPSwe/Ind mice. Biasettietal-iGluSnFRrationale: Background dataset used to establish iGluSnFR protocol used in the paper Biasettietal-abeta-time-conc-rationale: Supporting figure outlining the rationale for time and concentration used in this work. Abstract Alzheimer’s disease is linked to increased levels of amyloid beta (Aβ) in the brain, but the mechanisms underlying neuronal dysfunction and neurodegeneration remain enigmatic. Here, we investigate whether organizational characteristics of functional presynaptic vesicle pools, key determinants of information transmission in the central nervous system, are targets for elevated Aβ. Using an optical readout method in cultured hippocampal neurons, we show that acute Aβ42 treatment significantly enlarges the fraction of functional vesicles at individual terminals. We observe the same effect in a chronically elevated Aβ transgenic model (APPSw,Ind) using an ultrastructure-function approach that provides detailed information on nanoscale vesicle pool positioning. Strikingly, elevated Aβ is correlated with excessive accumulation of recycled vesicles near putative endocytic sites, which is consistent with deficits in vesicle retrieval pathways. Using the glutamate reporter, iGluSnFR, we show that there are parallel functional consequences, where ongoing information signaling capacity is constrained. Treatment with levetiracetam, an antiepileptic that dampens synaptic hyperactivity, partially rescues these transmission defects. Our findings implicate organizational and dynamic features of functional vesicle pools as targets in Aβ-driven synaptic impairment, suggesting that interventions to relieve the overloading of vesicle retrieval pathways might have promising therapeutic value.</p

A human proteogenomic-cellular framework identifies KIF5A as a modulator of astrocyte process integrity with relevance to ALS.

Genome-wide association studies identified several disease-causing mutations in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the contribution of genetic variants to pathway disturbances and their cell type-specific variations, especially in glia, is poorly understood. We integrated ALS GWAS-linked gene networks with human astrocyte-specific multi-omics datasets to elucidate pathognomonic signatures. It predicts that KIF5A, a motor protein kinesin-1 heavy-chain isoform, previously detected only in neurons, can also potentiate disease pathways in astrocytes. Using postmortem tissue and super-resolution structured illumination microscopy in cell-based perturbation platforms, we provide evidence that KIF5A is present in astrocyte processes and its deficiency disrupts structural integrity and mitochondrial transport. We show that this may underly cytoskeletal and trafficking changes in SOD1 ALS astrocytes characterised by low KIF5A levels, which can be rescued by c-Jun N-terminal Kinase-1 (JNK1), a kinesin transport regulator. Altogether, our pipeline reveals a mechanism controlling astrocyte process integrity, a pre-requisite for synapse maintenance and suggests a targetable loss-of-function in ALS

A human proteogenomic-cellular framework identifies KIF5A as a modulator of astrocyte process integrity with relevance to ALS

Acknowledgements: The authors are grateful to the funders. This project and the A.L. laboratory were funded by the Medical Research Council (UKRI MRC UK, MR/P008658/1; MR/X006867/1 to A.L.). Funding to support K.SZ. was provided by MRC UK (to A.L.), the IBRO Return Home Fellowship 2022 and the NKFIH/OTKA FK 142223 grant. I.B-H. was funded by Open Targets. We thank Matthew Gratian for his assistance with super-resolution microscopy, Dr András Füredi for providing MitoBright Red as a gift, and Professors Evan Reid, Stephen Sawcer, and Aviva Tolkovsky for their comments on our manuscript.Genome-wide association studies identified several disease-causing mutations in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the contribution of genetic variants to pathway disturbances and their cell type-specific variations, especially in glia, is poorly understood. We integrated ALS GWAS-linked gene networks with human astrocyte-specific multi-omics datasets to elucidate pathognomonic signatures. It predicts that KIF5A, a motor protein kinesin-1 heavy-chain isoform, previously detected only in neurons, can also potentiate disease pathways in astrocytes. Using postmortem tissue and super-resolution structured illumination microscopy in cell-based perturbation platforms, we provide evidence that KIF5A is present in astrocyte processes and its deficiency disrupts structural integrity and mitochondrial transport. We show that this may underly cytoskeletal and trafficking changes in SOD1 ALS astrocytes characterised by low KIF5A levels, which can be rescued by c-Jun N-terminal Kinase-1 (JNK1), a kinesin transport regulator. Altogether, our pipeline reveals a mechanism controlling astrocyte process integrity, a pre-requisite for synapse maintenance and suggests a targetable loss-of-function in ALS