Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Westmark, Pamela R.; Westmark, Cara J.; Jeevananthan, Athavi; Malter, James S.

doi:10.3791/3196

Cited by 25 publications

(27 citation statements)

References 29 publications

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“…We were able to entirely replicate these findings with 100 nM multimerized, soluble Aβ42 (fig. S2C) or with PiB, TAT-WW or juglone, all established Pin1 inhibitors (12) (Fig. 2D).…”

Section: Resultsmentioning

confidence: 92%

“…Neocortices from AD patients show progressive losses of Pin1 along with synapses and dendritic spines (11) that can be mimicked in cultured neurons (12) or slice preparations (13) treated with multimeric Aβ42. Unexpectedly, germ-line Pin1 KO cortex tissues exhibit increased dendritic spine density and increased LTP in hippocampal slices (6, 10).…”

Section: Resultsmentioning

confidence: 99%

“…Pin1 fl/fl mice in a C57Bl/6 background were used for all synaptoneurosome preparations per the method described in (12). Briefly, mouse cortices were harvested from mice that were heavily sedated with 2,2,2-tribromoethanol (Avertin).…”

Section: Methodsmentioning

confidence: 99%

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Pin1 mediates Aβ ₄₂ -induced dendritic spine loss

Stallings

O'Neal

et al. 2018

Sci. Signal.

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Early-stage Alzheimer’s disease is characterized by the loss of dendritic spines in the neocortex of the brain. This phenomenon precedes tau pathology, plaque formation, and neurodegeneration and likely contributes to synaptic loss, memory impairment, and behavioral changes in patients. Studies suggest that spine loss is induced by soluble, multimeric Aβ42, whose post-synaptic signaling activates the protein phosphatase calcineurin. We investigated how calcineurin causes spine pathology and found that the cis-trans prolyl isomerase Pin1 is a critical downstream target of Aβ42/calcineurin signaling. In spines, Pin1 interacts with and is dephosphorylated by calcineurin, which rapidly suppresses its isomerase activity. Pin1 knock-out or Aβ42 exposure induced mature spine loss that was prevented by exogenous Pin1. The calcinuerin inhibitor FK506 blocked spine loss in Aβ42-treated wild-type cells but had no effect on Pin1 null neurons. The data implicate Pin1 in spine maintenance and synaptic loss in early Alzheimer’s disease.

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“…We were able to entirely replicate these findings with 100 nM multimerized, soluble Aβ42 (fig. S2C) or with PiB, TAT-WW or juglone, all established Pin1 inhibitors (12) (Fig. 2D).…”

Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

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Pin1 mediates Aβ ₄₂ -induced dendritic spine loss

Stallings

O'Neal

et al. 2018

Sci. Signal.

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“…Three cortices (including hippocampi) per replicate were homogenized from Aldh1l1:EGFP/RPL10A mice and homogenate was spun at 1,000 × g for 10 min at 4°C, layered onto a sucrose-Percoll gradient as described previously (56), and spun at 32,500 × g for 5 min. The SN band was collected by puncturing the bottom of the tube to collect the SN fractions.…”

Section: Methodsmentioning

confidence: 99%

Astrocytes locally translate transcripts in their peripheral processes

Sakers

Lake

Khazanchi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

168

171

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Local translation in neuronal processes is key to the alteration of synaptic strength necessary for long-term potentiation, learning, and memory. Here, we present evidence that regulated de novo protein synthesis occurs within distal, perisynaptic astrocyte processes. Astrocyte ribosomal proteins are found adjacent to synapses in vivo, and immunofluorescent detection of peptide elongation in acute slices demonstrates robust translation in distal processes. We have also developed a biochemical approach to define candidate transcripts that are locally translated in astrocyte processes. Computational analyses indicate that astrocyte-localized translation is both sequence-dependent and enriched for particular biological functions, such as fatty acid synthesis, and for pathways consistent with known roles for astrocyte processes, such as GABA and glutamate metabolism. These transcripts also include glial regulators of synaptic refinement, such as Sparc. Finally, the transcripts contain a disproportionate amount of a binding motif for the quaking RNA binding protein, a sequence we show can significantly regulate mRNA localization and translation in the astrocytes. Overall, our observations raise the possibility that local production of astrocyte proteins may support microscale alterations of adjacent synapses.astrocyte | local translation | synapse | TRAP | RNA-sequencing

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“…Synaptoneurosomes used for in vitro DHPG stimulation experiments were isolated as previously reported (51). Synaptoneurosomes were equilibrated to room temperature for 10 min before stimulation with DHPG (100 μM).…”

Section: Methodsmentioning

confidence: 99%

Activity-dependent FUS dysregulation disrupts synaptic homeostasis

Sephton

Tang

Kulkarni

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

126

123

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The RNA-binding protein fused-in-sarcoma (FUS) has been associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), two neurodegenerative disorders that share similar clinical and pathological features. Both missense mutations and overexpression of wild-type FUS protein can be pathogenic in human patients. To study the molecular and cellular basis by which FUS mutations and overexpression cause disease, we generated novel transgenic mice globally expressing low levels of human wild-type protein (FUS WT ) and a pathological mutation (FUS R521G A myotrophic lateral sclerosis (ALS) is characterized by the degeneration of upper and lower motor neurons, leading to muscle weakness, paralysis, and death within 3-5 y of onset. Interestingly, ∼10-15% of ALS patients have clinical features of frontotemporal lobar degeneration (FTLD), marked by a decline in decision-making, behavioral control, emotion, and language, and as many as half have mild-to-moderate cognitive or behavioral abnormalities (1). FTLD comprises a group of heterogeneous diseases characterized by progressive neurodegeneration of the frontal and temporal lobes and clinically by frontotemporal dementia (FTD) with or without motor neuron disease. There is no cure or effective therapy for those who suffer from ALS or FTLD, and the mechanisms by which these diseases occur are not well understood.The clinical, pathological, and genetic overlap between ALS and FTLD suggests that there are mechanisms shared by these diseases. The RNA-binding proteins fused in sarcoma (FUS) and transactive response DNA-binding protein-43 (TDP-43) are the major protein components of inclusions that are characteristic of ALS and FTLD-U (FTLD with ubiquitinated inclusions) (2). More than 50 genetic FUS mutations have been identified in these related neurodegenerative disorders (3). Similarly, more than 40 dominant mutations in the TDP-43 gene have been linked to ALS cases and, to a lesser extent, to FTLD (4). The identification of mutations in the FUS and TDP-43 genes has provided insights for uncovering the disease mechanisms for ALS and FTLD.FUS is a ubiquitously expressed RNA-binding protein that exists in dynamic ribonucleoprotein complexes involved in pre-mRNA splicing, mRNA stability, and mRNA transport. FUS is a member of the FET family of proteins that bind RNAs (5) and contains an RNA recognition motif, three arginine-glycine-glycine (RGG) boxes, and a zinc finger (ZnF) (6). RGG2-ZnF-RGG3 is the major RNA-binding domain, which has a preference for GUrich sequences (7,8). The N terminus of FUS contains a lowcomplexity sequence domain involved in RNA granule formation (9). Nucleocytoplasmic shuttling of FUS occurs by a nonclassical proline-tyrosine nuclear localization signal (PY-NLS) and a nuclear export signal (NES) (10). Methylation of the C-terminal RGG3 domain of FUS is necessary for transportin 1 interaction and nuclear localization (11).The majority of clinical ALS/FTLD-associated FUS mutations occur in its C-terminal PY-NLS seque...

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Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Cited by 25 publications

References 29 publications

Pin1 mediates Aβ ₄₂ -induced dendritic spine loss

Pin1 mediates Aβ ₄₂ -induced dendritic spine loss

Astrocytes locally translate transcripts in their peripheral processes

Activity-dependent FUS dysregulation disrupts synaptic homeostasis

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Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Cited by 25 publications

References 29 publications

Pin1 mediates Aβ 42 -induced dendritic spine loss

Pin1 mediates Aβ 42 -induced dendritic spine loss

Astrocytes locally translate transcripts in their peripheral processes

Activity-dependent FUS dysregulation disrupts synaptic homeostasis

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Pin1 mediates Aβ ₄₂ -induced dendritic spine loss

Pin1 mediates Aβ ₄₂ -induced dendritic spine loss