Using Single-Molecule Fluorescence Microscopy to Uncover Neuronal Vulnerability to Protein Damage

Jacob-Tomas, Suleima; Boopathy, Lokha R Alagar; Vera, María

doi:10.1007/978-1-0716-2409-8_15

Cited by 4 publications

(10 citation statements)

References 35 publications

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“…To study how neurons tailor HSP expression to proteostatic demands, we subjected cultures of primary mouse hippocampal neurons to proteotoxic stress by inhibiting the proteasome. These cultures faithfully recapitulate the regulation of mRNA localization and translation in response to neuronal stimuli and the activation of HSP transcription upon stress 12 , 75 – 78 . Dissociated cultures of hippocampi from postnatal day 0 mouse pups differentiate to express features of their mature in situ counterparts by day 17.…”

Section: Resultsmentioning

confidence: 83%

Localized molecular chaperone synthesis maintains neuronal dendrite proteostasis

Ugalde,

Alecki,

Rizwan

et al. 2023

Preprint

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Proteostasis is maintained through regulated protein synthesis and degradation and chaperone-assisted protein folding. However, this is challenging in neuronal projections because of their polarized morphology and constant synaptic proteome remodeling. Using high-resolution fluorescence microscopy, we discovered that neurons localize a subset of chaperone mRNAs to their dendrites and use microtubule-based transport to increase this asymmetric localization following proteotoxic stress. The most abundant dendritic chaperone mRNA encodes a constitutive heat shock protein 70 family member (HSPA8). Proteotoxic stress also enhanced HSPA8 mRNA translation efficiency in dendrites. Stress-mediated HSPA8 mRNA localization to the dendrites was impaired by depleting fused in sarcoma—an amyotrophic lateral sclerosis-related protein—in cultured mouse motor neurons and expressing a pathogenic variant of heterogenous nuclear ribonucleoprotein A2/B1 in neurons derived from human induced pluripotent stem cells. These results reveal a crucial and unexpected neuronal stress response in which RNA-binding proteins increase the dendritic localization of HSPA8 mRNA to maintain proteostasis and prevent neurodegeneration.

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Section: Resultsmentioning

confidence: 83%

Localized molecular chaperone synthesis maintains neuronal dendrite proteostasis

Ugalde,

Alecki,

Rizwan

et al. 2023

Preprint

Self Cite

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show abstract

“…To study how neurons tailor the expression of HSPs to an increase in proteostasis demands, we subjected cultures of primary mouse hippocampal neurons to proteotoxic stress by inhibiting proteasomal activity. These cultures faithfully recapitulate the regulation of mRNA localization and translation in response to neuronal stimuli and the activation of HSP transcription upon stress ( 12 , 75–78 ). Dissociated cultures of hippocampi from postnatal day 0 mouse pups differentiate to express features of their mature in situ counterparts by day 17.…”

Section: Resultsmentioning

confidence: 86%

Localized synthesis of molecular chaperones sustains neuronal proteostasis

Alecki,

Rizwan,

et al. 2023

Preprint

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Neurons are challenged to maintain proteostasis in neuronal projections, particularly with the physiological stress at synapses to support intercellular communication underlying important functions such as memory and movement control. Proteostasis is maintained through regulated protein synthesis and degradation and chaperone-assisted protein folding. Using high-resolution fluorescent microscopy, we discovered that neurons localize a subset of chaperone mRNAs to their dendrites, particularly more proximal regions, and increase this asymmetric localization following proteotoxic stress through microtubule-based transport from the soma. The most abundant chaperone mRNA in dendrites encodes the constitutive heat shock protein 70, HSPA8. Proteotoxic stress in cultured neurons, induced by inhibiting proteasome activity or inducing oxidative stress, enhanced transport of Hspa8 mRNAs to dendrites and the percentage of mRNAs engaged in translation on mono and polyribosomes. Knocking down the ALS-related protein Fused in Sarcoma (FUS) and a dominant mutation in the heterogenous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) impaired stress-mediated localization of Hspa8 mRNA to dendrites in cultured murine motor neurons and human iPSC-derived neurons, respectively, revealing the importance of these RNA-binding proteins in maintaining proteostasis. These results reveal the increased dendritic localization and translation of the constitutive HSP70 Hspa8 mRNA as a crucial neuronal stress response to uphold proteostasis and prevent neurodegeneration.SUMMARYLocalizing chaperones’ mRNAs in neuronal dendrites is a novel on-demand system to uphold proteostasis upon stress.

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“…Motor neurons were microinjected with plasmids encoding the ALS variant and eGFP to identify injected cells. On day 1, 2, or 3 post microinjection, a combined smFISH with immunofluorescence protocol was followed as described in detail by Jacob-Tomas et al , 61 adapted from Eliscovich et al 62 The probes used for Mus musculus Hspa1a or Hspa8 mRNA (Stellaris LGC Biosearch Technologies, CA, USA) are a mixture of up to 48 lyophilized DNA oligonucleotides, each of them being between 18 and 22 nucleotides in length. 61 The sequence of probes labeled with Quasar 570 or Quasar 670 is presented in Supplementary Figure 1 .…”

Section: Methodsmentioning

confidence: 99%

“…The subcellular distribution of epifluorescent spots from the mRNA probe, representing single mRNAs, was analyzed using a specialized protocol developed by the Vera lab, employing a segmented computational approach using Python and FISH-Quant. 61 See Wefers et al . 63 for detailed methods.…”

Section: Methodsmentioning

confidence: 99%

Impact of histone deacetylase inhibition and arimoclomol on heat shock protein expression and disease biomarkers in primary culture models of familial ALS

Fernández Comaduran,

Minotti,

Jacob-Tomas

et al. 2024

Cell Stress and Chaperones

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Using Single-Molecule Fluorescence Microscopy to Uncover Neuronal Vulnerability to Protein Damage

Cited by 4 publications

References 35 publications

Localized molecular chaperone synthesis maintains neuronal dendrite proteostasis

Localized molecular chaperone synthesis maintains neuronal dendrite proteostasis

Localized synthesis of molecular chaperones sustains neuronal proteostasis

Impact of histone deacetylase inhibition and arimoclomol on heat shock protein expression and disease biomarkers in primary culture models of familial ALS

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