Synthesis and Characterization of Click Chemical Probes for Single-Cell Resolution Detection of Epichaperomes in Neurodegenerative Disorders.

Neurodegenerative disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD), represent debilitating conditions with complex, poorly understood pathologies. Epichaperomes, pathologic protein assemblies nucleated on key chaperones, have emerged as critical players in the molecular dysfunction underlying these disorders. In this study, we introduce the synthesis and characterization of clickable epichaperome probes, PU-TCO, positive control, and PU-NTCO, negative control. Through comprehensive in vitro assays and cell-based investigations, we establish the specificity of the PU-TCO probe for epichaperomes. Furthermore, we demonstrate the efficacy of PU-TCO in detecting epichaperomes in brain tissue with a cellular resolution, underscoring its potential as a valuable tool for dissecting single-cell responses in neurodegenerative diseases. This clickable probe is therefore poised to address a critical need in the field, offering unprecedented precision and versatility in studying epichaperomes and opening avenues for novel insights into their role in disease pathology.

Evaluation of Copper Chelation Therapy in a Transgenic Rat Model of Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA) is characterized by the accumulation of the amyloid ß (Aß) protein in blood vessels and leads to hemorrhages, strokes, and dementia in elderly individuals. Recent reports have shown elevated copper levels colocalized with vascular amyloid in human CAA and Alzheimer's disease patients, which have been suggested to contribute to cytotoxicity through the formation of reactive oxygen species. Here, we treated a transgenic rat model of CAA (rTg-DI) with the copper-specific chelator, tetrathiomolybdate (TTM), via intraperitoneal (IP) administration for 6 months to determine if it could lower copper content in vascular amyloid deposits and modify CAA pathology. Results showed that TTM treatment led to elevated Aß load in the hippocampus of the rTg-DI rats and increased microbleeds in the wild type (WT) animals. X-ray fluorescence microscopy was performed to image the distribution of copper and revealed a surprising increase in copper colocalized with Aß aggregates in TTM-treated rTg-DI rats. Unexpectedly, we also found an increase in the copper content in unaffected vessels of both rTg-DI and WT animals. These results show that IP administration of TTM was ineffective in removing copper from vascular Aß aggregates in vivo and increased the development of disease pathology in CAA.

Distinct Brain Proteomic Signatures in Cerebral Small Vessel Disease Rat Models of Hypertension and Cerebral Amyloid Angiopathy.

Cerebral small vessel diseases (CSVDs) are prominent contributors to vascular cognitive impairment and dementia and can arise from a range of etiologies. Cerebral amyloid angiopathy (CAA) and hypertension (HTN), both prevalent in the elderly population, lead to cerebral microhemorrhages, macrohemorrhages, and white matter damage. However, their respective underlying mechanisms and molecular events are poorly understood. Here, we show that the transgenic rat model of CAA type 1 (rTg-DI) exhibits perivascular inflammation that is lacking in the spontaneously hypertensive stroke-prone (SHR-SP) rat model of HTN. Alternatively, SHR-SP rats display notable dilation of arteriolar perivascular spaces. Comparative proteomics analysis revealed few shared altered proteins, with key proteins such as ANXA3, H2A, and HTRA1 unique to rTg-DI rats, and Nt5e, Flot-1 and Flot-2 unique to SHR-SP rats. Immunolabeling confirmed that upregulation of ANXA3, HTRA1, and neutrophil extracellular trap proteins were distinctly associated with rTg-DI rats. Pathway analysis predicted activation of TGF-ß1 and TNFα in rTg-DI rat brain, while insulin signaling was reduced in the SHR-SP rat brain. Thus, we report divergent protein signatures associated with distinct cerebral vessel pathologies in the SHR-SP and rTg-DI rat models and provide new mechanistic insight into these different forms of CSVD.

Impact of Non-pharmacological Chronic Hypertension on a Transgenic Rat Model of Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA), a common comorbidity of Alzheimer's disease (AD), is a cerebral small vessel disease (CSVD) characterized by deposition of fibrillar amyloid ß (Aß) in blood vessels of the brain and promotes neuroinflammation and vascular cognitive impairment and dementia (VCID). Hypertension, a prominent non-amyloidal CSVD, has been found to increase risk of dementia, but clinical data regarding its effects in CAA patients is controversial. To understand the effects of hypertension on CAA, we bred rTg-DI transgenic rats, a model of CAA, with spontaneously hypertensive, stroke prone (SHR-SP) rats producing bigenic rTg-DI/SHR-SP and non-transgenic SHR-SP littermates. At 7 months (M) of age, cohorts of both rTg-DI/SHR-SP and SHR-SP littermates exhibit elevated systolic blood pressures. However, transgene human amyloid ß-protein (Aß) precursor and Aß peptide levels, as well as behavioral testing showed no changes between bigenic rTg-DI/SHR-SP and rTg-DI rats. Subsequent cohorts of rats were aged further to 10 M where bigenic rTg-DI/SHR-SP and SHR-SP littermates exhibit elevated systolic and diastolic blood pressures. Vascular amyloid load in hippocampus and thalamus was significantly decreased, whereas pial surface vessel amyloid increased, in bigenic rTg-DI/SHR-SP rats compared to rTg-DI rats suggesting a redistribution of vascular amyloid in bigenic animals. There was activation of both astrocytes and microglia in rTg-DI rats and bigenic rTg-DI/SHR-SP rats not observed in SHR-SP rats indicating that glial activation was likely in response to the presence of vascular amyloid. Thalamic microbleeds were present in both rTg-DI rats and bigenic rTg-DI/SHR-SP rats. Although the number of thalamic small vessel occlusions were not different between rTg-DI and bigenic rTg-DI/SHR-SP rats, a significant difference in occlusion size and distribution in the thalamus was found. Proteomic analysis of cortical tissue indicated that bigenic rTg-DI/SHR-SP rats largely adopt features of the rTg-DI rats with enhancement of certain changes. Our findings indicate that at 10 M of age non-pharmacological hypertension in rTg-DI rats causes a redistribution of vascular amyloid and significantly alters the size and distribution of thalamic occluded vessels. In addition, our findings indicate that bigenic rTg-DI/SHR-SP rats provide a non-pharmacological model to further study hypertension and CAA as co-morbidities for CSVD and VCID.

Diffuse white matter loss in a transgenic rat model of cerebral amyloid angiopathy.

Diffuse white matter (WM) disease is highly prevalent in elderly with cerebral small vessel disease (cSVD). In humans, cSVD such as cerebral amyloid angiopathy (CAA) often coexists with Alzheimer's disease imposing a significant impediment for characterizing their distinct effects on WM. Here we studied the burden of age-related CAA pathology on WM disease in a novel transgenic rat model of CAA type 1 (rTg-DI). A cohort of rTg-DI and wild-type rats was scanned longitudinally using MRI for characterization of morphometry, cerebral microbleeds (CMB) and WM integrity. In rTg-DI rats, a distinct pattern of WM loss was observed at 9 M and 11 M. MRI also revealed manifestation of small CMB in thalamus at 6 M, which preceded WM loss and progressively enlarged until the moribund disease stage. Histology revealed myelin loss in the corpus callosum and thalamic CMB in all rTg-DI rats, the latter of which manifested in close proximity to occluded and calcified microvessels. The quantitation of CAA load in rTg-DI rats revealed that the most extensive microvascular Aß deposition occurred in the thalamus. For the first time using in vivo MRI, we show that CAA type 1 pathology alone is associated with a distinct pattern of WM loss.