Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution <em>In Situ</em> Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Columbia University

Columbia University Medical Center

Columbia College

We established and validated an in toto method to perform TdT-mediated dUTP nick end labeling to study apoptosis in human trabecular meshwork tissue obtained during trabeculectomy in glaucoma patients. In specimens from patients with primary open-angle glaucoma and primary angle-closure glaucoma, we detected a tendency for more apoptotic cells to accumulate in patients with primary open-angle glaucoma. The utility of this method to study apoptosis in the trabecular meshwork is discussed, as well as its application as a tool in biologic samples.

Apoptosis in the trabecular meshwork of glaucomatous patients.

Orchestrated proliferation, differentiation, and cell death contribute to the generation of the complex cytoarchitecture of the central nervous system, including that of the neuroretina. However, few studies have comprehensively compared the spatiotemporal patterns of these 3 processes, or their relative magnitudes. We performed a parallel study in embryonic chick and mouse retinas, focusing on the period during which the first neurons, the retinal ganglion cells (RGCs), are generated. The combination of in vivo BrdU incorporation, immunolabeling of retinal whole mounts for BrdU and for the neuronal markers Islet1/2 and β III-tubulin, and TUNEL allowed for precise cell scoring and determination the spatiotemporal patterns of cell proliferation, differentiation, and death. As predicted, proliferation preceded differentiation. Cell death and differentiation overlapped to a considerable extent, although the magnitude of cell death exceeded that of neuronal differentiation. Precise quantification of the population of recently born RGCs, identified by BrdU and β III-tubulin double labeling, combined with cell death inhibition using a pan-caspase inhibitor, revealed that apoptosis decreased this population by half shortly after birth. Taken together, our findings provide important insight into the relevance of cell death in neurogenesis.

Early neural cell death is an extensive, dynamic process in the embryonic chick and mouse retina.

In Alzheimer's disease (AD) brain, exposure of axons to Aβ causes pathogenic changes that spread retrogradely by unknown mechanisms, affecting the entire neuron. We found that locally applied Aβ1-42 initiates axonal synthesis of a defined set of proteins including the transcription factor ATF4. Inhibition of local translation and retrograde transport or knockdown of axonal Atf4 mRNA abolished Aβ-induced ATF4 transcriptional activity and cell loss. Aβ1-42 injection into the dentate gyrus (DG) of mice caused loss of forebrain neurons whose axons project to the DG. Protein synthesis and Atf4 mRNA were upregulated in these axons, and coinjection of Atf4 siRNA into the DG reduced the effects of Aβ1-42 in the forebrain. ATF4 protein and transcripts were found with greater frequency in axons in the brain of AD patients. These results reveal an active role for intra-axonal translation in neurodegeneration and identify ATF4 as a mediator for the spread of AD pathology.

Axonally synthesized ATF4 transmits a neurodegenerative signal across brain regions.

Localized protein synthesis is a mechanism by which morphologically polarized cells react in a spatially confined and temporally acute manner to changes in their environment. During the development of the nervous system intra-axonal protein synthesis is crucial for the establishment of neuronal connections. In contrast, mature axons have long been considered as translationally inactive but upon nerve injury or under neurodegenerative conditions specific subsets of mRNAs are recruited into axons and locally translated. Intra-axonally synthesized proteins can have pathogenic or restorative and regenerative functions, and thus targeting the axonal translatome might have therapeutic value, for example in the treatment of spinal cord injury or Alzheimer's disease. In the case of Alzheimer's disease the local synthesis of the stress response transcription factor activating transcription factor 4 mediates the long-range retrograde spread of pathology across the brain, and inhibition of local Atf4 translation downstream of the integrated stress response might interfere with this spread. Several molecular tools and approaches have been developed to target specifically the axonal translatome by either overexposing proteins locally in axons or, conversely, knocking down selectively axonally localized mRNAs. Many questions about axonal translation remain to be answered, especially with regard to the mechanisms establishing specificity but, nevertheless, targeting the axonal translatome is a promising novel avenue to pursue in the development for future therapies for various neurological conditions.

Jimena Baleriola

College of Physicians and Surgeons

Columbia University

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Jimena Baleriola

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Columbia University

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

College of Physicians and Surgeons