Microglial and Astrocytic Responses in the Human Midcingulate Cortex in Huntington's Disease.

OBJECTIVE: Patients with Huntington's disease can present with variable difficulties of motor functioning, mood, and cognition. Neurodegeneration occurs in the anterior cingulate cortex of some patients with Huntington's disease and is linked to the presentation of mood symptomatology. Neuroinflammation, perpetrated by activated microglia and astrocytes, has been reported in Huntington's disease and may contribute to disease progression and presentation. This study sought to quantify the density of mutant huntingtin protein and neuroinflammatory glial changes in the midcingulate cortex of postmortem patients with Huntington's disease and determine if either correlates with the presentation of mood, motor, or mixed symptomatology. METHODS: Free-floating immunohistochemistry quantified 1C2 immunolabeling density as an indicative marker of mutant huntingtin protein, and protein and morphological markers of astrocyte (EAAT2, Cx43, and GFAP), and microglial (Iba1 and HLA-DP/DQ/DR) activation. Relationships among the level of microglial activation, mutant huntingtin burden, and case characteristics were explored using correlative analysis. RESULTS: We report alterations in activated microglia number and morphology in the midcingulate cortex of Huntington's disease cases with predominant mood symptomatology. An increased proportion of activated microglia was observed in the midcingulate of all Huntington's disease cases and positively correlated with 1C2 burden. Alterations in the astrocytic glutamate transporter EAAT2 were observed in the midcingulate cortex of patients associated with mood symptoms. INTERPRETATION: This study presents pathological changes in microglia and astrocytes in the midcingulate cortex in Huntington's disease, which coincide with mood symptom presentation. These findings further the understanding of neuroinflammation in Huntington's disease, a necessary step for developing inflammation-targeted therapeutics. ANN NEUROL 2023;94:895-910.

Normal aging, motor neurone disease, and Alzheimer's disease are characterized by cortical changes in inflammatory cytokines.

The role of increased brain inflammation in the development of neurodegenerative diseases is unclear. Here, we have compared cytokine changes in normal aging, motor neurone disease (MND), and Alzheimer's disease (AD). After an initial analysis, six candidate cytokines, interleukin (IL)- 4, 5, 6, 10, macrophage inhibitory protein (MIP)-1α, and fibroblast growth factor (FGF)-2, showing greatest changes were assayed in postmortem frozen human superior frontal gyri (n = 12) of AD patients, aging and young adult controls along with the precentral gyrus (n = 12) of MND patients. Healthy aging was associated with decreased anti-inflammatory IL-10 and FGF-2 levels. AD prefrontal cortex was associated with increased levels of IL-4, IL-5, and FGF-2, with the largest increase seen for FGF-2. Notwithstanding differences in the specific frontal lobe gyrus sampled, MND patients' primary motor cortex (precentral gyrus) was associated with increased levels of IL-5, IL-6, IL-10, and FGF-2 compared to the aging prefrontal cortex (superior frontal gyrus). Immunocytochemistry showed that FGF-2 is expressed in neurons, astrocytes, and microglia in normal aging prefrontal cortex, AD prefrontal cortex, and MND motor cortex. We report that healthy aging and age-related neurodegenerative diseases have different cortical inflammatory signatures that are characterized by increased levels of anti-inflammatory cytokines and call into question the view that increased inflammation underlies the development of age-related neurodegenerative diseases.

The effect of age and sex on the expression of GABA signaling components in the human hippocampus and entorhinal cortex.

Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the nervous system. The GABA signaling system in the brain is comprised of GABA synthesizing enzymes, transporters, GABAA and GABAB receptors (GABAAR and GABABR). Alterations in the expression of these signaling components have been observed in several brain regions throughout aging and between sexes in various animal models. The hippocampus is the memory centre of the brain and is impaired in several age-related disorders. It is composed of two main regions: the Cornu Ammonis (CA1-4) and the Dentate Gyrus (DG), which are interconnected with the Entorhinal Cortex (ECx). The age- and sex-specific changes of GABA signaling components in these regions of the human brain have not been examined. This study is the first to determine the effect of age and sex on the expression of GABA signaling components-GABAAR α1,2,3,5, ß1-3, γ2, GABABR R1 and R2 subunits and the GABA synthesizing enzymes GAD 65/67-in the ECx, and the CA1 and DG regions of the human hippocampus using Western blotting. No significant differences were found in GABAAR α1,2,3,5, ß1-3, γ2, GABABR R1 and R2 subunit and GAD65/76 expression levels in the ECx, CA1 and DG regions between the younger and older age groups for both sexes. However, we observed a significant negative correlation between age and GABAAR α1subunit level in the CA1 region for females; significant negative correlation between age and GABAAR ß1, ß3 and γ2 subunit expression in the DG region for males. In females a significant positive correlation was found between age and GABAAR γ2 subunit expression in the ECx and GABABR R2 subunit expression in the CA1 region. The results indicate that age and sex do not affect the expression of GAD 65/67. In conclusion, our results show age- and sex-related GABAA/BR subunit alterations in the ECx and hippocampus that might significantly influence GABAergic neurotransmission and underlie disease susceptibility and progression.

Huntington's disease mice and human brain tissue exhibit increased G3BP1 granules and TDP43 mislocalization.

Chronic cellular stress associated with neurodegenerative disease can result in the persistence of stress granule (SG) structures, membraneless organelles that form in response to cellular stress. In Huntington's disease (HD), chronic expression of mutant huntingtin generates various forms of cellular stress, including activation of the unfolded protein response and oxidative stress. However, it has yet to be determined whether SGs are a feature of HD neuropathology. We examined the miRNA composition of extracellular vesicles (EVs) present in the cerebrospinal fluid (CSF) of patients with HD and show that a subset of their target mRNAs were differentially expressed in the prefrontal cortex. Of these targets, SG components were enriched, including the SG-nucleating Ras GTPase-activating protein-binding protein 1 (G3BP1). We investigated localization and levels of G3BP1 and found a significant increase in the density of G3BP1-positive granules in the cortex and hippocampus of R6/2 transgenic mice and in the superior frontal cortex of the brains of patients with HD. Intriguingly, we also observed that the SG-associated TAR DNA-binding protein 43 (TDP43), a nuclear RNA/DNA binding protein, was mislocalized to the cytoplasm of G3BP1 granule-positive HD cortical neurons. These findings suggest that G3BP1 SG dynamics may play a role in the pathophysiology of HD.

The Interplay Between Beta-Amyloid 1-42 (Aß1-42)-Induced Hippocampal Inflammatory Response, p-tau, Vascular Pathology, and Their Synergistic Contributions to Neuronal Death and Behavioral Deficits.

Alzheimer's disease (AD), the most common chronic neurodegenerative disorder, has complex neuropathology. The principal neuropathological hallmarks of the disease are the deposition of extracellular ß-amyloid (Aß) plaques and neurofibrillary tangles (NFTs) comprised of hyperphosphorylated tau (p-tau) protein. These changes occur with neuroinflammation, a compromised blood-brain barrier (BBB) integrity, and neuronal synaptic dysfunction, all of which ultimately lead to neuronal cell loss and cognitive deficits in AD. Aß1-42 was stereotaxically administered bilaterally into the CA1 region of the hippocampi of 18-month-old male C57BL/6 mice. This study aimed to characterize, utilizing immunohistochemistry and behavioral testing, the spatial and temporal effects of Aß1-42 on a broad set of parameters characteristic of AD: p-tau, neuroinflammation, vascular pathology, pyramidal cell survival, and behavior. Three days after Aß1-42 injection and before significant neuronal cell loss was detected, acute neuroinflammatory and vascular responses were observed. These responses included the up-regulation of glial fibrillary acidic protein (GFAP), cell adhesion molecule-1 (PECAM-1, also known as CD31), fibrinogen labeling, and an increased number of activated astrocytes and microglia in the CA1 region of the hippocampus. From day 7, there was significant pyramidal cell loss in the CA1 region of the hippocampus, and by 30 days, significant localized up-regulation of p-tau, GFAP, Iba-1, CD31, and alpha-smooth muscle actin (α-SMA) in the Aß1-42-injected mice compared with controls. These molecular changes in Aß1-42-injected mice were accompanied by cognitive deterioration, as demonstrated by long-term spatial memory impairment. This study is reporting a comprehensive examination of a complex set of parameters associated with intrahippocampal administration of Aß1-42 in mice, their spatiotemporal interactions and combined contribution to the disease progression. We show that a single Aß injection can reproduce aspects of the inflammatory, vascular, and p-tau induced pathology occurring in the AD human brain that lead to cognitive deficits.

An 5 GABAA Receptor Inverse Agonist, 5IA, Attenuates Amyloid Beta-Induced Neuronal Death in Mouse Hippocampal Cultures.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder for which no cognition-restoring therapies exist. Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain. Increasing evidence suggests a remodeling of the GABAergic system in AD, which might represent an important therapeutic target. An inverse agonist of 5 subunit-containing GABAA receptors (α5GABAARs), 3-(5-Methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-yl)methyloxy]-1,2,4-triazolo[3-a]phthalazine (5IA) has cognition-enhancing properties. This study aimed to characterize the effects of 5IA on amyloid beta (A1-42)-induced molecular and cellular changes. Mouse primary hippocampal cultures were exposed to either A1-42 alone, or 5IA alone, 5IA with A1-42 or vehicle alone, and changes in cell viability and mRNA expression of several GABAergic signaling components were assessed. Treatment with 100 nM of 5IA reduced A1-42-induced cell loss by 23.8% (p < 0.0001) after 6 h and by 17.3% after 5 days of treatment (p < 0.0001). Furthermore, we observed an A1-42-induced increase in ambient GABA levels, as well as upregulated mRNA expression of the GABAAR α2,α5,2/3 subunits and the GABABR R1 and R2 subunits. Such changes in GABARs expression could potentially disrupt inhibitory neurotransmission and normal network activity. Treatment with 5IA restored A1-42-induced changes in the expression of α5GABAARs. In summary, this compound might hold neuroprotective potential and represent a new therapeutic avenue for AD.

GABAA and GABAB receptor subunit localization on neurochemically identified neurons of the human subthalamic nucleus.

The subthalamic nucleus (STN) is a critical excitatory signaling center within the basal ganglia circuitry. The activity of subthalamic neurons is tightly controlled by upstream inhibitory signaling centers in the basal ganglia. In this study, we used immunohistochemical techniques to firstly, visualize and quantify the STN neurochemical organization based on neuronal markers including parvalbumin (PV), calretinin (CR), SMI-32, and GAD65/67 . Secondly, we characterized the detailed regional, cellular and subcellular expression of GABAA (α1 , α2 , α3 , ß2/3 , and γ2 ) and GABAB (R1 and R2) receptor subunits within the normal human STN. Overall, we found seven neurochemically distinct populations of principal neurons in the human STN. The three main populations detected were: (a) triple-labeled PV+ /CR+ /SMI32+ ; (b) double-labeled PV+ /CR+ ; and (c) single-labeled CR+ neurons. Subthalamic principal neurons were found to express GABAA receptor subunits α1 , α3 , ß2/3 , γ2 , and GABAB receptor subunits R1 and R2. However, no expression of GABAA receptor α2 subunit was detected. We also found a trend of increasing regional staining intensity for all positive GABAA receptor subunits from the dorsolateral pole to ventromedial extremities. The GAD+ interneurons showed relatively low expression of GABAA receptor subunits. These results provide the morphological basis of GABAergic transmission within the normal human subthalamic nucleus and evidence of GABA innervation through both GABAA and GABAB receptors on subthalamic principal neurons.