ABSTRACT Apolipoprotein E (ApoE) is a major genetic risk factor for sporadic Alzheimer's disease and a potential therapeutic target, but the relative contribution of peripheral blood‐derived and intrathecally synthesized ApoE to the CNS remains unclear. A novel electrophoresis‐based capillary Western blot assay was validated to quantify Pan‐ApoE and ApoE4 in paired human CSF and serum samples ( n = 34), followed by analysis of CSF/serum quotients. Serum ApoE concentrations were ninefold higher than CSF concentrations, with CSF/serum quotients of 0.132 for Pan‐ApoE and 0.191 for ApoE4. Compared with theoretical quotients predicted for proteins entering CSF solely by passive diffusion according to their hydrodynamic radius according to Felgenhauer et al., the observed quotients were 66‐ to 1320‐fold higher for Pan‐ApoE and 96‐ to 1910‐fold higher for ApoE4. Based on this model, passive diffusion was estimated to account for less than 1.5% of CSF ApoE. These estimates are model‐dependent and may vary with ApoE isoform and extent of lipidation, which could potentially be affected by serum fibrin clot formation. The remaining ApoE fraction (> 98.5%) may reflect active transport, intrathecal synthesis or both. Pan‐ApoE and ApoE4 CSF/serum quotients lacked statistically significant association with CSF/serum albumin quotient as a marker of blood‐CSF barrier function. Both quotients also exceeded the values predicted by a theoretical reference curve relating CSF/serum protein quotients to the albumin quotient, adapted from the Reiber model using immunoglobulin G parameters based on the comparable hydrodynamic radii of IgG and ApoE. image
ABSTRACT Inhibition of glycogen synthase kinase‐3 (GSK‐3) has been implicated in alleviating Huntington's disease (HD)‐associated phenotypes. However, the mechanisms linking GSK‐3 to HD pathogenesis, and particularly the distinct roles of GSK‐3 isozymes in regulating HD‐associated cellular dysfunction, remain poorly understood. Here, we investigated the cellular responses of patient‐derived HD fibroblasts to selective inhibition of GSK‐3α or GSK‐3β using isozyme‐specific inhibitors, focusing on metabolic and bioenergetic pathways. Although inhibition of either isozyme suppressed mTORC1 signaling, selective GSK‐3β inhibition elicited broader cellular responses, including altered oxidative stress signaling, increased expression of proteins associated with mitochondrial fission, enhanced lysosomal activity, and mitophagy, together with lysosomal redistribution and increased α‐tubulin acetylation, suggesting improved microtubule‐dependent intracellular trafficking. Quantitative proteomic analysis of four independent patient‐derived HD fibroblasts corroborated these findings: GSK‐3β inhibition elicited a broad set of proteins involved in cytoskeletal organization, adhesion signaling, and metabolic adaptation, whereas GSK‐3α inhibition produced a more restricted and heterogeneous response. Collectively, these findings identify distinct cellular responses to selective GSK‐3 isozyme inhibition and reveal GSK‐3β as a predominant regulator of intracellular trafficking and mitochondrial and cytoskeletal dynamics in HD fibroblasts. Thus, inhibition of GSK‐3β may represent a potential therapeutic approach for modulating HD‐associated cellular dysfunction. image
ABSTRACT Down syndrome (DS) is a genetic aneuploidy characterized by a variable phenotype that includes a certain degree of intellectual disability. These alterations have been associated with impaired cell proliferation, reduced structural plasticity, and imbalances in inhibitory‐excitatory neurotransmission. Adults with DS exhibit increased inhibition, whereas fetuses and newborns display deficits in inhibitory signaling. In this study, we confirmed that new‐born Ts65Dn mice, the most widely used model of DS, show a reduction in inhibitory synapses. We treated Ts65Dn pups daily for 4 weeks after birth with tiagabine, a GABA transporter 1 inhibitor that increases extracellular GABA levels, in an attempt to restore normal inhibition. This early intervention restored the total number of synapses and normalized the excess of inhibitory contacts previously described in the adult Ts65Dn hippocampus. The expression of plasticity‐related molecules, such as PSA‐NCAM, increased in parallel with the recovery of spine density on granule cells and thorny excrescences on CA3 pyramidal neurons. Moreover, cell proliferation, neurogenesis, and cellular activity were restored to control levels. Overall, these results indicate that early intervention targeting GABAergic neurotransmission during a critical developmental window may be an effective strategy to reverse hippocampal alterations in Ts65Dn mice and suggest that GAT‐1 may have therapeutic potential for individuals with DS. image
ABSTRACT Rab GTPases are key regulators of membrane trafficking. However, few Rabs have been identified to participate in the secretion processes of synaptic and secretory vesicles. In the present study, we sought to investigate the role of Rab11a, a protein classically involved in recycling pathways, in the regulation of dense‐core vesicle (DCV) secretion in mouse chromaffin cells, a well‐established neurosecretory model. By the use of confocal imaging, we found that Rab11a colocalized with DCV. Importantly, the expression of the dominant‐negative mutant (S25N) or constitutive‐active mutant (Q70L) forms of Rab11a increased the DCV cargo neuropeptide Y (NPY) at Golgi. Additionally, upon expression of S25N or Q70L, or the overexpression of Rab11a‐WT form, we found that: (1) NPY decreased at the cell periphery; (2) the fluorescent timer mK‐GO‐NPY showed a decrease in newly formed DCV at the cell periphery; and (3) there was a decrease in the exocytic response, measured by patch‐clamped membrane capacitance recording. Remarkably, the effects of Rab11a‐S25N or Rab11a‐Q70L expression were stronger than those observed upon Rab11a‐WT overexpression, suggesting a dependency of DCV distribution and secretion on Rab11a GDP‐to‐GTP cycle. On the other hand, the effects of Rab11a‐WT overexpression may be explained by an imbalance toward the GTP‐bound form of Rab11a, which might sequester endogenous effectors and freeze the exocytic process at intermediate steps. Therefore, this study suggests that Rab11a facilitates the supply of DCV at the plasma membrane by positively modulating the generation and/or transport of new DCV, and, in this way, supporting the exocytic response. image
