The neuroendocrine peptide somatostatin (SST) has long been thought of as influencing the deposition of the amyloid β peptide (Aβ) in Alzheimer’s disease (AD). Missing have been in vivo data in a relevant Aβ amyloidosis model. Here we crossed App NL-F/NL-F mice with Sst -deficient mice to assess if and how the presence of Sst influences pathological hallmarks of Aβ amyloidosis. We found that Sst had no influence on whole brain neprilysin transcript, protein or activity levels, an observation that cannot be accounted for by a compensatory upregulation of the Sst paralog, cortistatin (Cort), that we observed in 15-month-old Sst-deficient mice. Sst-deficiency led to a subtle but significant increase in the density of cortical Aβ amyloid plaques. Follow-on western blot analyses of whole brain extracts indicated that Sst interferes with early steps of Aβ assembly that manifest in the appearance of SDS-stable smears of 55–150 kDa in Sst null brain samples. As expected, no effect of Sst on tau steady-state levels or its phosphorylation were observed. Results from this study are easier reconciled with an emerging body of data that point toward Sst affecting Aβ amyloid plaque formation through direct interference with Aβ aggregation rather than through its effects on neprilysin expression.

ABSTRACTThe molecular underpinnings that govern the endoproteolytic release of the amyloid beta peptide (Aβ) from the amyloid precursor protein (APP) are now quite well understood. The same cannot be said for the events that precipitate the aggregation and amyloid deposition of Aβ in Alzheimer’s disease (AD). The 14-amino-acid cyclic neuroendocrine peptide somatostatin (SST-14) has long been thought of as playing a role, foremost by controlling the expression of the Aβ clearing enzyme neprilysin, and more recently by directly interacting with Aβ oligomers. Missing have been in vivo data in a relevant Aβ amyloidosis model. Here we addressed this shortcoming by crossing AppNL-F/NL-F mice with Sst-deficient mice of identical genetic background to assess if and how the presence of Sst influences key pathological hallmarks of Aβ amyloidosis that develop in AppNL-F/NL-F mice after 10 months of age. Surprisingly, we found that Sst had no influence on whole brain neprilysin transcript, protein or activity levels, an observation that cannot be accounted for by a compensatory upregulation of the Sst paralog, cortistatin (Cort), that we observed in 15-month-old Sst-deficient mice. The absence of Sst did lead to a subtle but significant increase in the density of cortical Aβ amyloid plaques. Follow-on western blot analyses of whole brain extracts indicated that Sst interferes with early steps of Aβ assembly that manifest in Sst null brains through the appearance of SDS-stable smears of 55- 150 kDa. As expected, no effect of Sst on tau steady-state levels or its phosphorylation were observed. Results from this study are easier reconciled with an emerging body of data that point toward Sst affecting Aβ amyloid plaque formation through direct interference with Aβ aggregation rather than through its effects on neprilysin expression.

It is widely anticipated that a reduction of brain levels of the cellular prion protein (PrPC) can prolong survival in a group of neurodegenerative diseases known as prion diseases. To date, efforts to decrease steady-state PrPC levels by targeting this protein directly with small molecule drug-like compounds have largely been unsuccessful. Recently, we reported Na,K-ATPases to reside in immediate proximity to PrPC in the brain, unlocking an opportunity for an indirect PrPC targeting approach that capitalizes on the availability of potent cardiac glycosides (CGs). Here, we report that exposure of human co-cultures of neurons and astrocytes to non-toxic nanomolar levels of CGs causes profound reductions in PrPC levels. The mechanism of action underpinning this outcome relies primarily on a subset of CGs engaging the ATP1A1 isoform, one of three α subunits of Na,K-ATPases expressed in brain cells. Upon CG docking to ATP1A1, the ligand receptor complex, and PrPC along with it, is internalized by the cell. Subsequently, PrPC is channeled to the lysosomal compartment where it is digested in a manner that can be rescued by silencing the cysteine protease cathepsin B. These data signify that the repurposing of CGs may be beneficial for the treatment of prion disorders.

The prion protein (PrP) is best known for its ability to cause fatal neurodegenerative diseases in humans and animals. Here, we revisited its molecular environment in the brain using a well-developed affinity-capture mass spectrometry workflow that offers robust relative quantitation. The analysis confirmed many previously reported interactions. It also pointed toward a profound enrichment of Na,K-ATPases (NKAs) in proximity to cellular PrP (PrP C ). Follow-on work validated the interaction, demonstrated partial co-localization of the ATP1A1 and PrP C , and revealed that cells exposed to cardiac glycoside (CG) inhibitors of NKAs exhibit correlated changes to the steady-state levels of both proteins. Moreover, the presence of PrP C was observed to promote the ion uptake activity of NKAs in a human co-culture paradigm of differentiated neurons and glia cells, and in mouse neuroblastoma cells. Consistent with this finding, changes in the expression of 5’-nucleotidase that manifest in wild-type cells in response to CG exposure can also be observed in untreated PrP C -deficient cells. Finally, the endoproteolytic cleavage of the glial fibrillary acidic protein, a hallmark of late-stage prion disease, can also be induced by CGs, raising the prospect that a loss of NKA activity may contribute to the pathobiology of prion diseases.

The misfolding of transactive response DNA-binding protein (TDP-43) is a major contributor to the pathogenesis of TDP-43 proteinopathies, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 inclusions, but also plays a role in other neurodegenerative diseases including Alzheimer disease. It is thought that different truncations at the N- and C-termini of TDP-43 contribute to its misfolding and aggregation in the brain, and that these aberrant TDP-43 fragments contribute to disease. Despite this, little is known about whether different truncation events influence the protein's transmissibility between cells and how this cell-to-cell transfer occurs. In this study, we use a well-established cellular model to study the efficiency by which full-length and truncated TDP-43 fragments are transferred between neuron-like cells. We demonstrate that preservation of the N-terminus of TDP-43 enhances its transmissibility between cells and that this protein transmission occurs in a manner exclusive of extracellular vesicles, instead requiring cellular proximity for efficient propagation. These data indicate that the N-terminus of TDP-43 might be a useful target in the generation of therapeutics to limit the spread of TDP-43 pathology.

Alzheimer’s disease (AD) is the most common form of dementia globally and is characterized by aberrant accumulations of amyloid-beta (Aβ) and tau proteins. Oligomeric forms of these proteins are believed to be most relevant to disease progression, with oligomeric amyloid-β (oAβ) particularly implicated in AD. oAβ pathology spreads among interconnected brain regions, but how oAβ induces pathology in these previously unaffected neurons requires further study. Here, we use well characterized iPSC-derived human neurons to study the early changes to the proteome and phosphoproteome after 24 h exposure to oAβ 1-42. Using nLC-MS/MS and label-free quantification, we identified several proteins that are differentially regulated in response to acute oAβ challenge. At this early timepoint, oAβ induced the decrease of TDP-43, heterogeneous nuclear ribonucleoproteins (hnRNPs), and coatomer complex I (COPI) proteins. Conversely, increases were observed in 20 S proteasome subunits and vesicle associated proteins VAMP1/2, as well as the differential phosphorylation of tau at serine 208. These changes show that there are widespread alterations to the neuronal proteome within 24 h of oAβ uptake, including proteins previously not shown to be related to neurodegeneration. This study provides new targets for the further study of early mediators of AD pathogenesis.

Recently, extracellular vesicles (EVs), such as exosomes, have been proposed to play an influential role in the cell-to-cell spread of neurodegenerative diseases, including the intercellular transmission of α-synuclein (α-syn). However, the regulation of EV biogenesis and its relation to Parkinson’s disease (PD) is only partially understood. The generation of EVs through the ESCRT-independent pathway depends on the hydrolysis of sphingomyelin by neutral sphingomyelinase 2 (nSMase2) to produce ceramide, which causes the membrane of endosomal multivesicular bodies to bud inward. nSMase2 is sensitive to oxidative stress, a common process in PD brains; however, little is known about the role of sphingomyelin metabolism in the pathogenesis of PD. This is the first study to show that inhibiting nSMase2 decreases the transfer of oligomeric aggregates of α-syn between neuron-like cells. Furthermore, it reduced the accumulation and aggregation of high-molecular-weight α-syn. Hypoxia, as a model of oxidative stress, reduced the levels of nSMase2, but not its enzymatic activity, and significantly altered the lipid composition of cells without affecting EV abundance or the transfer of α-syn. These data show that altering sphingolipids can mitigate the spread of α-syn, even under hypoxic conditions, potentially suppressing PD progression.

The intercellular transfer of alpha-synuclein (α-syn) has been implicated in the progression of Parkinson’s disease (PD) and multiple system atrophy (MSA). The cellular mechanisms underlying this process are now beginning to be elucidated. In this study, we demonstrate that the gap junction protein connexin-32 (Cx32) is centrally involved in the preferential uptake of α-syn oligomeric assemblies (oα-syn) in neurons and oligodendrocytes. In vitro, we demonstrate a clear correlation between Cx32 expression and oα-syn uptake. Pharmacological and genetic strategies targeting Cx32 successfully blocked oα-syn uptake. In cellular and transgenic mice modeling PD and MSA, we observed significant upregulation of Cx32 which correlates with α-syn accumulation. Notably, we could also demonstrate a direct interaction between α-syn and Cx32 in two out of four human PD cases that was absent in all four age-matched controls. These data are suggestive of a link between Cx32 and PD pathophysiology. Collectively, our results provide compelling evidence for Cx32 as a novel target for therapeutic intervention in PD and related α-synucleinopathies.

Neuroinflammation plays an influential role in Alzheimer's disease (AD), although the mechanisms underlying this phenomenon remain largely unknown. Microglia are thought to be responsible for the majority of these effects and can be characterized into resting (M0), proinflammatory (M1), or anti-inflammatory (M2) functional phenotypes. We investigated the effects of conditioned macrophage media, as an analogue to microglia, on the transfer of oligomeric amyloid beta (oAβ) between differentiated SH-SY5Y cells. We also investigated how the different inflammatory environments related to intercellular and intracellular changes. We demonstrate that M2 products decrease interneuronal transfer of oAβ, while recombinant interleukin (IL)-4, IL-10, and IL-13 increase transfer. There were no alterations to the mRNA of a number of AD-related genes in response to the combination of oAβ and M0, M1, or M2, but several intracellular proteins, some relating to protein trafficking and the endosomal/lysosomal system, were altered. Stimulating microglia to an M2 phenotype may thus slow down the progression of AD and could be a target for future therapies.

Parkinson’s disease and other alpha-synucleinopathies are progressive neurodegenerative diseases characterized by aggregates of misfolded alpha-synuclein spreading throughout the brain. Recent evidence suggests that the pathological progression is likely due to neuron-to-neuron transfer of these aggregates between neuroanatomically connected areas of the brain. As the impact of this pathological spreading mechanism is currently debated, we aimed to investigate the transfer and subcellular location of alpha-synuclein species in a novel 3D co-culture human cell model based on highly differentiated SH-SY5Y cells. Fluorescently-labeled monomeric, oligomeric and fibrillar species of alpha-synuclein were introduced into a donor cell population and co-cultured with an EGFP-expressing acceptor-cell population of differentiated neuron-like cells. Subsequent transfer and colocalization of the different species were determined with confocal microscopy. We could confirm cell-to-cell transfer of all three alpha-synuclein species investigated. Interestingly the level of transferred oligomers and fibrils and oligomers were significantly higher than monomers, which could affect the probability of seeding and pathology in the recipient cells. Most alpha-synuclein colocalized with the lysosomal/endosomal system, both pre- and postsynaptically, suggesting its importance in the processing and spreading of alpha-synuclein.

The SH-SY5Y-cell line is widely used as an in-vitro model for Alzheimer's disease, and there is evidence that SH-SY5Y cells can have an electrophysiological behavior similar to neurons. It has been suggested that SH-SY5Y cells are capable of having a depolarizing current mediated by sodium channels and hyperpolarizing currents induced by potassium channels and could trigger action potentials. Potentially, these cells may be able to create a synaptic structure and form a neuronal-like network. We have previously shown that cell-to-cell transfer of β-amyloid depends on existing neuritic connections. The aim this work was to investigate how SH-SY5Y cells change their electrical activity under varying levels of differentiation and how neurodegenerative proteins affect this activity. SH-SY5Y cells were untreated, differentiated with retinoic acid or differentiated with retinoic acid in addition to a cocktail of growth factors resulting in a highly neuronal-like morphology. The electrophysiological properties were tested in both cell-attached and whole-cell patch clamp configuration plus current-clamp or voltage clamp. Subsequently, the electrophysiological properties after treatment with neurodegenerative proteins (oligomers of β-amyloid or α -synuclein) were tested. We recorded scarce spontaneous electrical activity in untreated SH-SY5Y cells, and observed an increase in spontaneous activity in the presence of cellular connections, particularly in differentiated cells. This indicates that SH-SY5Y cells form functional synapses in relation to differentiation. This shows the feasibility to use differentiated SH-SY5Y cells to investigate the electrophysiological effects of propagation of neurodegenerative proteins. We show that SH-SY5Y cells differentiated with a cocktail of growth factors have a neuronal behavior beyond morphology and expression of neuronal markers. We propose that differentiated SH-SY5Y cells could be used to assess how neurodegenerative proteins could modify the synaptic function during disease propagation throughout the neuronal network.

The progression of Alzheimer's disease and Parkinson's disease follows a hierarchical anatomical pattern. Recent evidence indicates that this could be caused by a prion-like spread of the disease with aggregates of β-amyloid and α-synuclein respectively, propagating from neuron-to-neuron. It is known that α-synuclein oligomers can transfer between cells but not if this requires neurites and synapses. We have previously visualized neuritic neuron-to-neuron transfer of β-amyloid, in an 3D co-culture system with differentiated neuron-like human SH-SY5Y cells. In this study we investigated if α-synuclein is transferred in a similar manner as β-amyloid. We also investigated the intracellular localization and toxicity of the transferred α-synuclein. Monomeric, oligomeric and fibrillar forms of a -synuclein were labeled with Cy3. SH-SY5Y neuroblastoma cells were extensively differentiated in an ECM gel with the addition of a cocktail of growth-factors and transfected with CellLight® endosome-GFP (acceptor cells). Retinoic acid differentiated SH-SY5Y cells were incubated with 1-2 μM monomeric, oligomeric or fibrillary α-synuclein for 3 h (donor cells). Donor cells and acceptor cells were co-cultured for up to 7 days, fixed and analyzed using confocal microscopy. Monomeric, oligomeric and fibrillar α-synuclein forms were taken up by donor cells within 3 hours and remained in the cells for at least 48 hours. After 24 h of co-culture, all tested forms of α-synuclein were transferred from donor cells to acceptor cells if neuritic connections were present. Oligomers were transferred more extensively than monomers. The α-synuclein co-localized with markers for multi vesicular bodies, endosoms and lysosomes. Monomeric, oligomeric and fibrillar forms of α-synuclein were transmitted through neuritic connections from donor to acceptor cells. The transfer of α-synuclein seem to involve multivesicular bodies as well as the endosomal and lysosomal compartments. The high level of differentiation of the acceptor cells, producing neuritic connections and synapses, seemed necessary for the transfer. Thus, we have found many similarities between α-synuclein and β-amyloid transfer.