γ-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter, playing a central role in the regulation of cortical excitability and the maintenance of the excitatory/inhibitory (E/I) balance. Several lines of evidence point to a remodeling of the cerebral GABAergic system in Alzheimer’s disease (AD), with past studies demonstrating alterations in GABA receptor and transporter expression, GABA synthesizing enzyme activity and focal GABA concentrations in post-mortem tissue. AD is a chronic neurodegenerative disorder with a poorly understood etiology and the temporal cortex is one of the earliest regions in the brain to be affected by AD neurodegeneration. Utilizing NanoString nCounter analysis, we demonstrate here the transcriptional downregulation of several GABA signaling components in the post-mortem human middle temporal gyrus (MTG) in AD, including the GABA_A receptor α₁, α₂, α₃, α₅, β₁, β₂, β₃, δ, γ₂, γ₃, and θ subunits and the GABA_B receptor 2 (GABA_BR2) subunit. In addition to this, we note the transcriptional upregulation of the betaine-GABA transporter (BGT1) and GABA transporter 2 (GAT2), and the downregulation of the 67 kDa isoform of glutamate decarboxylase (GAD₆₇), the primary GABA synthesizing enzyme. The functional consequences of these changes require further investigation, but such alterations may underlie disruptions to the E/I balance that are believed to contribute to cognitive decline in AD.

Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the nervous system. Previous studies have shown fluctuations in expression levels of GABA signaling components—glutamic acid decarboxylase (GAD), GABA receptor (GABAR) subunit, and GABA transporter (GAT)—with increasing age and between sexes; however, this limited knowledge is highly based on animal models that produce inconsistent findings. This study is the first analysis of the age- and sex-specific changes of the GAD, GABAA/BR subunits, and GAT expression in the human primary sensory and motor cortices; superior (STG), middle (MTG), and inferior temporal gyrus (ITG); and cerebellum. Utilizing Western blotting, we found that the GABAergic system is relatively robust against sex and age-related differences in all brain regions examined. However, we observed several sex-dependent differences in GABAAR subunit expression in STG along with age-dependent GABAAR subunit and GAD level alteration. No significant age-related differences were found in α1, α2, α5, β3, and γ2 subunit expression in the STG. However, we found significantly higher GABAAR α3 subunit expression in the STG in young males compared to old males. We observed a significant sex-dependent difference in α1 subunit expression: males presenting significantly higher levels compared to women across all stages of life in STG. Older females showed significantly lower α2, α5, and β3 subunit expression compared to old males in the STG. These changes found in the STG might significantly influence GABAergic neurotransmission and lead to sex- and age-specific disease susceptibility and progression.

In Alzheimer’s disease (AD), microglia are affected by disease processes, but may also drive pathogenesis. AD pathology-associated microglial populations have been identified with single-cell RNA-Seq, but have not been validated in human brain tissue with anatomical context. Here, we quantified myeloid cell markers to identify changes in AD pathology-associated microglial populations. We performed fluorescent immunohistochemistry on normal (n = 8) and AD (n = 8) middle temporal gyri, co-labelling the pan-myeloid cell marker, Iba1, with one of 11 markers of interest (MOIs): CD45, HLA-DR, CD14, CD74, CD33, CD206, CD32, CD163, P2RY12, TMEM119, L-Ferritin. Novel image analyses quantified the single-cell abundance of Iba1 and each MOI. Each cell was gated into one Iba1-MOI population (Iba1^low MOI^high, Iba1^high MOI^high, or Iba1^high MOI^low) and the abundance of each population was compared between AD and control. Triple-labelling of L-Ferritin and Iba1 with a subset of MOIs was performed to investigate L-Ferritin-MOI co-expression on Iba1^low cells. Iba1^low MOI^high myeloid cell populations delineated by MOIs CD45, HLA-DR, CD14, CD74, CD33, CD32, and L-Ferritin were increased in AD. Further investigation of the Iba1^low MOI^high populations revealed that their abundances correlated with tau, but not amyloid beta, load in AD. The Iba1^low microglial population highly expressed L-Ferritin, reflecting microglial dysfunction. The L-Ferritin^high CD74^high HLA-DR^high phenotype of the Iba1^low population mirrors that of a human AD pathology-associated microglial subpopulation previously identified using single-cell RNA-Seq. Our high-throughput immunohistochemical data with anatomical context support the microglial dysfunction hypothesis of AD.

In Parkinson’s disease (PD), and other α-synucleinopathies, α-synuclein (α-Syn) aggregates form a myriad of conformational and truncational variants. Most antibodies used to detect and quantify α-Syn in the human brain target epitopes within the C-terminus (residues 96–140) of the 140 amino acid protein and may fail to capture the diversity of α-Syn variants present in PD. We sought to investigate the heterogeneity of α-Syn conformations and aggregation states in the PD human brain by labelling with multiple antibodies that detect epitopes along the entire length of α-Syn. We used multiplex immunohistochemistry to simultaneously immunolabel tissue sections with antibodies mapping the three structural domains of α-Syn. Discrete epitope-specific immunoreactivities were visualised and quantified in the olfactory bulb, medulla, substantia nigra, hippocampus, entorhinal cortex, middle temporal gyrus, and middle frontal gyrus of ten PD cases, and the middle temporal gyrus of 23 PD, and 24 neurologically normal cases. Distinct Lewy neurite and Lewy body aggregate morphologies were detected across all interrogated regions/cases. Lewy neurites were the most prominent in the olfactory bulb and hippocampus, while the substantia nigra, medulla and cortical regions showed a mixture of Lewy neurites and Lewy bodies. Importantly, unique N-terminus immunoreactivity revealed previously uncharacterised populations of (1) perinuclear, (2) glial (microglial and astrocytic), and (3) neuronal lysosomal α-Syn aggregates. These epitope-specific N-terminus immunoreactive aggregate populations were susceptible to proteolysis via time-dependent proteinase K digestion, suggesting a less stable oligomeric aggregation state. Our identification of unique N-terminus immunoreactive α-Syn aggregates adds to the emerging paradigm that α-Syn pathology is more abundant and complex in human brains with PD than previously realised. Our findings highlight that labelling multiple regions of the α-Syn protein is necessary to investigate the full spectrum of α-Syn pathology and prompt further investigation into the functional role of these N-terminus polymorphs.

Impairment of brain-glucose uptake and brain-copper regulation occurs in Alzheimer’s disease (AD). Here we sought to further elucidate the processes that cause neurodegeneration in AD by measuring levels of metabolites and metals in brain regions that undergo different degrees of damage. We employed mass spectrometry (MS) to measure metabolites and metals in seven post-mortem brain regions of nine AD patients and nine controls, and plasma-glucose and plasma-copper levels in an ante-mortem case-control study. Glucose, sorbitol and fructose were markedly elevated in all AD brain regions, whereas copper was correspondingly deficient throughout (all P < 0.0001). In the ante-mortem case-control study, by contrast, plasma-glucose and plasma-copper levels did not differ between patients and controls. There were pervasive defects in regulation of glucose and copper in AD brain but no evidence for corresponding systemic abnormalities in plasma. Elevation of brain glucose and deficient brain copper potentially contribute to the pathogenesis of neurodegeneration in AD.