# The Functional Epididymal Amyloid Cystatin‐Related Epididymal Spermatogenic (CRES) is a Component of the Mammalian Brain Extracellular Matrix

## TL;DR

The CRES protein, known for its role in the male reproductive system, is found in the brain's extracellular matrix and may function as an amyloid to support brain structure and function.

## Contribution

This study identifies CRES as a novel amyloid component of the brain extracellular matrix with potential roles in brain function.

## Key findings

- CRES is produced by hippocampal neurons and astrocytes in both male and female mouse and human brains.
- CRES levels decline in aged mice astrocytes, potentially contributing to altered brain function.
- CRES colocalizes with ECM markers and exists in amyloid forms in brain regions like the hippocampus and cortex.

## Abstract

CRES is the defining member of a reproductive subgroup of family 2 cystatins of cysteine protease inhibitors. We previously showed that CRES and other subgroup members are part of a highly plastic amyloid‐containing extracellular matrix (ECM) with host defense functions in the mouse epididymal lumen. Based on parallels between the epididymis and the brain, we hypothesized that CRES and CRES amyloids might also function within the brain including the ECM. Here we show that CRES is produced by hippocampal neurons and astrocytes in the male and female mouse and human brain. Further, approximately 50% of hippocampal astrocytes from aged mice, like the aged human donor samples, had significantly reduced levels of CRES compared to younger mice, suggesting an age‐related decline in CRES could contribute to altered brain function. Immunofluorescence experiments showed CRES colocalized with the ECM markers phosphacan and
wisteria floribunda
agglutinin indicating that CRES is part of the ECM. CRES monomer and high molecular weight SDS‐resistant forms were found in insoluble fractions of the hippocampus, cortex, cerebellum, and midbrain and bound to the protein aggregation disease (PAD) ligand, which preferentially binds amyloids but not protein monomers, suggesting a population of CRES normally exists in the brain as an amyloid structure. Collectively, our studies demonstrate that CRES/CRES amyloid is present in the mammalian brain and may contribute to ECM structure and function.

The cystatin‐related epididymal spermatogenic (CRES) protein contributes to an amyloid‐containing extracellular matrix (ECM) that protects the male germline in the epididymal lumen. Using cell biological and biochemical approaches, we show here that CRES is found in hippocampal astrocytes and specific neuronal populations, including those that possess perineuronal nets, and is colocalized with ECM markers suggesting it is part of the ECM. Biochemical analyses suggested a population of CRES is present as an amyloid structure. Our studies reveal CRES is present in the male and female mammalian brain and may contribute to brain structure and function as a biological amyloid.

This study investigated if the epididymal amyloid CRES (cystatin‐related epididymal spermatogenic) is present in the male and female mammalian brain. CRES is expressed by hippocampal astrocytes, CA2 and CA3 neurons, and exhibits amyloid properties in the extracellular matrix. These data suggest CRES contributes to brain ECM function as a biological amyloid.