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Cultured hippocampal neurons glial fibrillary acidic protein (GFAP)

ABSTRACT: Despite their widespread use as a research model, a comprehensive, quantitative proteomic profile of the cultured hippocampal neurons has remained unexplored. Here, we provide the first global proteomic characterization of primary murine hippocampal neurons cultured for 14 days under near-physiological glucose conditions (2.5 mM). Using label-free mass spectrometry and the Total Protein Approach (TPA), we quantified over 5,500 proteins, revealing a metabolic signature consistent with oxidative phosphorylation and a neuron-specific proteome largely devoid of astrocytic markers. Unexpectedly, glial fibrillary acidic protein (GFAP) was highly abundant in pure neuronal cultures, and its knockdown impaired neuronal viability and morphology. These findings suggest a compensatory role for GFAP in neurons cultured without astrocytic support. Additionally, our data highlight significant underexpression of key synaptic proteins compared to hippocampal tissue, underscoring the limitations of neuron-only culture systems. This study establishes a new molecular reference for hippocampal neurons and offers insights into neuronal adaptation in simplified environments.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Positive Regulation Of Neuron Apoptotic Process, Cell Culture

SUBMITTER: Mariusz Fleszar

LAB HEAD: Mariusz Fleszar

PROVIDER: PXD067465 | Pride | 2026-01-22

REPOSITORIES: Pride

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			Action	DRS
	2025_05_13_RAKUS_PROTEO_CALE_KOMORKI_PIII_50FMOL_Hi_01.mzML	Mzml
	2025_05_13_RAKUS_PROTEO_CALE_KOMORKI_PIII_50FMOL_Hi_02.mzML	Mzml
	2025_05_13_RAKUS_PROTEO_CALE_KOMORKI_PIII_50FMOL_Hi_03.mzML	Mzml
	2025_05_13_RAKUS_PROTEO_CALE_KOMORKI_PII_50FMOL_Hi_01.mgf	Mgf
	2025_05_13_RAKUS_PROTEO_CALE_KOMORKI_PII_50FMOL_Hi_01.mzML	Mzml

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Project description:Progranulin is a secreted pro-protein that is trafficked to lysosomes and exerts protective effects in the brain. Progranulin (GRN) mutations, most of which cause progranulin haploinsufficiency, are a major genetic cause of frontotemporal dementia (FTD). Restoring progranulin to people with GRN mutations is a promising treatment strategy, but understanding progranulin’s mechanism of action may enable design of optimal progranulin-based therapies. Progranulin restrains inflammation and promotes neuronal growth and survival, but the mechanisms underlying these effects are unclear. Progranulin is constitutively secreted and interacts with various signaling receptors, but is also taken up and trafficked to lysosomes, where it is necessary for maintaining normal lysosomal function. In previous work, we showed that progranulin acts in lysosomes to promote neuronal survival, but it is not clear if progranulin enhances neuronal growth by acting in lysosomes or through extracellular signaling. To address this question, we employed lentiviral vectors expressing either progranulin (PGRN) or a non-secreted, lysosome-targeted progranulin (L-PGRN). Using lentiviral vectors driven by non-selective (PGK), neuron-selective (hSyn), or astrocyte-selective (GFAP) promoters, we found that delivering L-PGRN to astrocytes, but not neurons, promoted dendritic outgrowth in primary hippocampal cultures. Using transwell co-cultures of astrocytes and neurons, we found that neurons cultured with L-PGRN–transduced astrocytes had greater dendritic outgrowth than those cultured with GFP-transduced astrocytes. Bulk RNA sequencing of primary astrocytes indicated that L-PGRN reduced transcriptomic pathways associated with inflammation and cellular reactivity. Consistent with this result, we found that reducing the number of astrocytes in hippocampal cultures increased dendritic outgrowth and occluded the pro-growth effects of L-PGRN. These data show that under these culture conditions, progranulin secretion is not required to promote dendritic outgrowth. Instead, progranulin may act via a non-cell–autonomous mechanism to suppress secretion of factors from astrocytes that restrain neuronal growth. These data add to a growing body of evidence that progranulin can act on astrocytes to promote neuronal health.

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Cultured hippocampal neurons glial fibrillary acidic protein (GFAP)

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