Alzheimer's Disease-associated Region-specific Decrease of Vesicular Glutamate Transporter Immunoreactivity inthe Medial Temporal Lobe and Superior Temporal Gyrus.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder for which there are very limited treatment options. Dysfunction of the excitatory neurotransmitter system is thought to play a major role in the pathogenesis of this condition. Vesicular glutamate transporters (VGLUTs) are key to controlling the quantal release of glutamate. Thus, expressional changes in disease can have implications for aberrant neuronal activity, raising the possibility of a therapeutic target. There is no information regarding the expression of VGLUTs in the human medial temporal lobe in AD, one of the earliest and most severely affected brain regions. This study aimed to quantify and compare the layer-specific expression of VGLUT1 and VGLUT2 between control and AD cases in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus. Free-floating fluorescent immunohistochemistry was used to label VGLUT1 and VGLUT2 in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus. Sections were imaged using laser-scanning confocal microscopy and transporter densitometric analysis was performed. VGLUT1 density was not significantly different in AD tissue, except lower staining density observed in the dentate gyrus stratum moleculare (p = 0.0051). VGLUT2 expression was not altered in the hippocampus and entorhinal cortex of AD cases but was significantly lower in the subiculum (p = 0.015) and superior temporal gyrus (p = 0.0023). This study indicates a regionally specific vulnerability of VGLUT1 and VGLUT2 expression in the medial temporal lobe and superior temporal gyrus in AD. However, the causes and functional consequences of these disturbances need to be further explored to assess VGLUT1 and VGLUT2 as viable therapeutic targets.

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.

Current and Possible Future Therapeutic Options for Huntington's Disease.

Huntington's disease (HD) is an autosomal neurodegenerative disease that is characterized by an excessive number of CAG trinucleotide repeats within the huntingtin gene (HTT). HD patients can present with a variety of symptoms including chorea, behavioural and psychiatric abnormalities and cognitive decline. Each patient has a unique combination of symptoms, and although these can be managed using a range of medications and non-drug treatments there is currently no cure for the disease. Current therapies prescribed for HD can be categorized by the symptom they treat. These categories include chorea medication, antipsychotic medication, antidepressants, mood stabilizing medication as well as non-drug therapies. Fortunately, there are also many new HD therapeutics currently undergoing clinical trials that target the disease at its origin; lowering the levels of mutant huntingtin protein (mHTT). Currently, much attention is being directed to antisense oligonucleotide (ASO) therapies, which bind to pre-RNA or mRNA and can alter protein expression via RNA degradation, blocking translation or splice modulation. Other potential therapies in clinical development include RNA interference (RNAi) therapies, RNA targeting small molecule therapies, stem cell therapies, antibody therapies, non-RNA targeting small molecule therapies and neuroinflammation targeted therapies. Potential therapies in pre-clinical development include Zinc-Finger Protein (ZFP) therapies, transcription activator-like effector nuclease (TALEN) therapies and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) therapies. This comprehensive review aims to discuss the efficacy of current HD treatments and explore the clinical trial progress of emerging potential HD therapeutics.

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.

EAAT2 Expression in the Hippocampus, Subiculum, Entorhinal Cortex and Superior Temporal Gyrus in Alzheimer's Disease.

Alzheimer's disease (AD) is a neuropathological disorder characterized by the presence and accumulation of amyloid-beta plaques and neurofibrillary tangles. Glutamate dysregulation and the concept of glutamatergic excitotoxicity have been frequently described in the pathogenesis of a variety of neurodegenerative disorders and are postulated to play a major role in the progression of AD. In particular, alterations in homeostatic mechanisms, such as glutamate uptake, have been implicated in AD. An association with excitatory amino acid transporter 2 (EAAT2), the main glutamate uptake transporter, dysfunction has also been described. Several animal and few human studies examined EAAT2 expression in multiple brain regions in AD but studies of the hippocampus, the most severely affected brain region, are scarce. Therefore, this study aims to assess alterations in the expression of EAAT2 qualitatively and quantitatively through DAB immunohistochemistry (IHC) and immunofluorescence within the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus (STG) regions, between human AD and control cases. Although no significant EAAT2 density changes were observed between control and AD cases, there appeared to be increased transporter expression most likely localized to fine astrocytic branches in the neuropil as seen on both DAB IHC and immunofluorescence. Therefore, individual astrocytes are not outlined by EAAT2 staining and are not easily recognizable in the CA1-3 and dentate gyrus regions of AD cases, but the altered expression patterns observed between AD and control hippocampal cases could indicate alterations in glutamate recycling and potentially disturbed glutamatergic homeostasis. In conclusion, no significant EAAT2 density changes were found between control and AD cases, but the observed spatial differences in transporter expression and their functional significance will have to be further explored.

Glutamatergic receptor expression changes in the Alzheimer's disease hippocampus and entorhinal cortex.

Alzheimer's Disease (AD) is the leading form of dementia worldwide. Currently, the pathological mechanisms underlying AD are not well understood. Although the glutamatergic system is extensively implicated in its pathophysiology, there is a gap in knowledge regarding the expression of glutamate receptors in the AD brain. This study aimed to characterize the expression of specific glutamate receptor subunits in post-mortem human brain tissue using immunohistochemistry and confocal microscopy. Free-floating immunohistochemistry and confocal laser scanning microscopy were used to quantify the density of glutamate receptor subunits GluA2, GluN1, and GluN2A in specific cell layers of the hippocampal sub-regions, subiculum, entorhinal cortex, and superior temporal gyrus. Quantification of GluA2 expression in human post-mortem hippocampus revealed a significant increase in the stratum (str.) moleculare of the dentate gyrus (DG) in AD compared with control. Increased GluN1 receptor expression was found in the str. moleculare and hilus of the DG, str. oriens of the CA2 and CA3, str. pyramidale of the CA2, and str. radiatum of the CA1, CA2, and CA3 subregions and the entorhinal cortex. GluN2A expression was significantly increased in AD compared with control in the str. oriens, str. pyramidale, and str. radiatum of the CA1 subregion. These findings indicate that the expression of glutamatergic receptor subunits shows brain region-specific changes in AD, suggesting possible pathological receptor functioning. These results provide evidence of specific glutamatergic receptor subunit changes in the AD hippocampus and entorhinal cortex, indicating the requirement for further research to elucidate the pathophysiological mechanisms it entails, and further highlight the potential of glutamatergic receptor subunits as therapeutic targets.

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.

Amyloid-beta1-42 induced glutamatergic receptor and transporter expression changes in the mouse hippocampus.

Alzheimer's disease (AD) is the leading type of dementia worldwide. With an increasing burden of an aging population coupled with the lack of any foreseeable cure, AD warrants the current intense research effort on the toxic effects of an increased concentration of beta-amyloid (Aß) in the brain. Glutamate is the main excitatory brain neurotransmitter and it plays an essential role in the function and health of neurons and neuronal excitability. While previous studies have shown alterations in expression of glutamatergic signaling components in AD, the underlying mechanisms of these changes are not well understood. This is the first comprehensive anatomical study to characterize the subregion- and cell layer-specific long-term effect of Aß1-42 on the expression of specific glutamate receptors and transporters in the mouse hippocampus, using immunohistochemistry with confocal microscopy. Outcomes are examined 30 days after Aß1-42 stereotactic injection in aged male C57BL/6 mice. We report significant decreases in density of the glutamate receptor subunit GluA1 and the vesicular glutamate transporter (VGluT) 1 in the conus ammonis 1 region of the hippocampus in the Aß1-42 injected mice compared with artificial cerebrospinal fluid injected and naïve controls, notably in the stratum oriens and stratum radiatum. GluA1 subunit density also decreased within the dentate gyrus dorsal stratum moleculare in Aß1-42 injected mice compared with artificial cerebrospinal fluid injected controls. These changes are consistent with findings previously reported in the human AD hippocampus. By contrast, glutamate receptor subunits GluA2, GluN1, GluN2A, and VGluT2 showed no changes in expression. These findings indicate that Aß1-42 induces brain region and layer specific expression changes of the glutamatergic receptors and transporters, suggesting complex and spatial vulnerability of this pathway during development of AD neuropathology. Read the Editorial Highlight for this article on page 7. Cover Image for this issue: https://doi.org/10.1111/jnc.14763.

The Role of Microglia and Astrocytes in Huntington's Disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease. HD patients present with movement disorders, behavioral and psychiatric symptoms and cognitive decline. This review summarizes the contribution of microglia and astrocytes to HD pathophysiology. Neuroinflammation in the HD brain is characterized by a reactive morphology in these glial cells. Microglia and astrocytes are critical in regulating neuronal activity and maintaining an optimal milieu for neuronal function. Previous studies provide evidence that activated microglia and reactive astrocytes contribute to HD pathology through transcriptional activation of pro-inflammatory genes to perpetuate a chronic inflammatory state. Reactive astrocytes also display functional changes in glutamate and ion homeostasis and energy metabolism. Astrocytic and microglial changes may further contribute to the neuronal death observed with the progression of HD. Importantly, the degree to which these neuroinflammatory changes are detrimental to neurons and contribute to the progression of HD pathology is not well understood. Furthermore, recent observations provide compelling evidence that activated microglia and astrocytes exert a variety of beneficial functions that are essential for limiting tissue damage and preserving neuronal function in the HD brain. Therefore, a better understanding of the neuroinflammatory environment in the brain in HD may lead to the development of targeted and innovative therapeutic opportunities.

GABAA Receptors Are Well Preserved in the Hippocampus of Aged Mice.

GABA is the primary inhibitory neurotransmitter in the nervous system. GABAA receptors (GABAARs) are pentameric ionotropic channels. Subunit composition of the receptors is associated with the affinity of GABA binding and its downstream inhibitory actions. Fluctuations in subunit expression levels with increasing age have been demonstrated in animal and human studies. However, our knowledge regarding the age-related hippocampal GABAAR expression changes is limited and based on rat studies. This study is the first analysis of the aging-related changes of the GABAAR subunit expression in the CA1, CA2/3, and dentate gyrus regions of the mouse hippocampus. Using Western blotting and immunohistochemistry we found that the GABAergic system is robust, with no significant age-related differences in GABAAR α1, α2, α3, α5, ß3, and γ2 subunit expression level differences found between the young (6 months) and old (21 months) age groups in any of the hippocampal regions examined. However, we detected a localized decrease of α2 subunit expression around the soma, proximal dendrites, and in the axon initial segment of pyramidal cells in the CA1 and CA3 regions that is accompanied by a pronounced upregulation of the α2 subunit immunoreactivity in the neuropil of aged mice. In summary, GABAARs are well preserved in the mouse hippocampus during normal aging although GABAARs in the hippocampus are severely affected in age-related neurological disorders, including Alzheimer's disease.

Sex- and age-related changes in GABA signaling components in the human cortex.

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, ß3, 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 ß3 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.

The Acute Effects of Amyloid-Beta1-42 on Glutamatergic Receptor and Transporter Expression in the Mouse Hippocampus.

Alzheimer's disease (AD) is the leading type of dementia worldwide. Despite an increasing burden of disease due to a rapidly aging population, there is still a lack of complete understanding of the precise pathological mechanisms which drive its progression. Glutamate is the main excitatory neurotransmitter in the brain and plays an essential role in the normal function and excitability of neuronal networks. While previous studies have shown alterations in the function of the glutamatergic system in AD, the underlying etiology of beta amyloid (Aß1-42) induced changes has not been explored. Here we have investigated the acute effects of stereotaxic hippocampal Aß1-42 injection on specific glutamatergic receptors and transporters in the mouse hippocampus, using immunohistochemistry and confocal microscopy 3 days after Aß1-42 injection in aged male C57BL/6 mice, before the onset of neuronal cell death. We show that acute injection of Aß1-42 is sufficient to induce cognitive deficits 3 days post-injection. We also report no significant changes in glutamate receptor subunits GluA1, GluA2, VGluT1, and VGluT2 in response to acute injection of Aß1-42 when compared with the ACSF-vehicle injected mice. However, we observed increased expression in the DG hilus and ventral stratum (str.) granulosum, CA3 str. radiatum and str. oriens, and CA1 str. radiatum of the GluN1 subunit, and increased expression within the CA3 str. radiatum and decreased expression within the DG str. granulosum of the GluN2A subunit in Aß1-42 injected mice compared to NC, and a similar trend observed when compared to ACSF-injected mice. We also observed alterations in expression patterns of glutamatergic receptor subunits and transporters within specific layers of hippocampal subregions in response to a microinjection stimulus. These findings indicate that the pathological alterations in the glutamatergic system observed in AD are likely to be partially a result of both acute and chronic exposure to Aß1-42 and implies a much more complex circuit mechanism associated with glutamatergic dysfunction than simply glutamate-mediated excitotoxic neuronal death.

Impaired expression of GABA transporters in the human Alzheimer's disease hippocampus, subiculum, entorhinal cortex and superior temporal gyrus.

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain and plays an important role in regulating neuronal excitability. GABA reuptake from the synapse is dependent on specific transporters - mainly GAT-1, GAT-3 and BGT-1 (GATs). This study is the first to show alterations in the expression of the GATs in the Alzheimer's disease (AD) hippocampus, entorhinal cortex and superior temporal gyrus. We found a significant increase in BGT-1 expression associated with AD in all layers of the dentate gyrus, in the stratum oriens of the CA2 and CA3 and the superior temporal gyrus. In AD there was a significant decrease in GAT-1 expression in the entorhinal cortex and superior temporal gyrus. We also found a significant decrease in GAT-3 immunoreactivity in the stratum pyramidale of the CA1 and CA3, the subiculum and entorhinal cortex. These observations indicate that the expression of the GATs shows brain-region- and layer-specific alterations in AD, suggesting a complex activation pattern of different GATs during the course of the disease.