Glial response in the midcingulate cortex in Huntington’s disease

Huntington’s disease (HD) is a genetic disease characterized by the progressive degeneration of the striatum and cortex. Patients can present with a variety of symptoms that can broadly be classified into motor symptoms, inclusive of choreatic movements and rigidity, mood and psychiatric symptoms, such as depression and apathy, and cognitive symptoms, such as cognitive decline. The causal mutation underlying HD results from an expansion of a CAG repeat sequence on the IT15 gene, resulting in the formation and accumulation of a mutant huntingtin protein.

The definitive mechanisms underlying the progressive atrophy of the striatum and cortex are yet to be elucidated, however, the potential contribution of a neuroinflammatory glial response in exacerbating cell death has been postulated (Palpagama et al., 2019). The gene expression and protein changes in astrocytes and microglial cells can result in the initiation and perpetuation of an inflammatory response. Chronic inflammation can, in turn promote the release of reactive oxygen species, proinflammatory cytokines, acidic species, and dysregulate ion and neurotransmitter balances (Palpagama et al., 2019). Indeed, there is an implication of astrocytic involvement in HD with advancing astrocytosis in the striatum considered a defining hallmark of the disease progression. Further to this, activated microglia, referring to microglia expressing genes and proteins that result in the deviation of these cells from a resting state, were observed in the striatum and frontal cortex with a correlative relationship between the number of activated microglia and the extent of neuronal loss (Sapp et al., 2001). Together, these studies suggest a significant glial response concomitant to cell death.

Atrophy of specific loci of the brain has been postulated to underlie the development of specific symptom profiles by patients. The progressive degeneration of the striatum has been long thought to underlie motor symptoms such as chorea and rigidity. Further research has highlighted the contribution of the atrophy of cortical regions to the development of different symptom profiles. This is exemplified by stereological cell counting studies that have identified pyramidal neuronal losses in the motor cortex, superior frontal, superior parietal, mid-temporal, primary sensory, and secondary visual cortices in brain tissue from patients with mainly motor symptoms when compared with controls. Interestingly, tissue from patients with mainly mood symptomology exhibited pyramidal neuron losses in the anterior cingulate cortex, superior frontal cortex, middle temporal, and superior parietal cortices (Thu et al., 2010; Nana et al., 2014). From this seminal work, the hypothesis that the development of a mood-predominant symptomology profile may be the result of the progressive deterioration of the anterior cingulate cortex (ACC) and associational areas of the frontal, middle, and parietal cortices that contribute to mood, has developed. The association between the regional atrophy of the brain in a symptom-specific way coupled with the potential contribution of glial cells through the development of a neurotoxic environment suggests the importance of investigating the glial response in pathologically affected areas of the HD brain.

The cingulate cortex is a region of particular interest when studying the deterioration of the cortex in HD due to the connectivity of this structure to multiple brain regions. The immense connectivity of the cingulate underlies the involvement of this structure in many functions including emotion processing, cognition, nociceptive and visuospatial functions, autonomic regulation, and skeletomotor functions. The ACC presents a role in emotion processing, forming part of the limbic system and presenting extensive connectivity to limbic structures such as the amygdala. Therefore, it is not surprising that the degeneration of this area has been correlated to the development of mood symptom profiles. Interestingly, the examination of microglia in the ACC suggests the presence of reactive microglia in magnetic resonance imaging studies (Pavese et al., 2006). Astrocytes were also detected to show altered gene expression patterns in the ACC in HD that were consistent with reactive phenotypes. Taken together, these studies suggest that in the ACC, glial responses are occurring and may relate to the symptom-specific atrophy in this area (Al-Dalahmah et al., 2020). Interestingly, the neighboring midcingulate cortex (MCC) however is far less characterized than the ACC. Stereological counting of a region of Brodmann area 24, which forms a portion of the MCC, revealed no changes in neuronal cell number in HD (Macdonald and Halliday, 2002). However, it is critical to note this study did not investigate anatomical changes across all Brodmann areas that form the MCC. Magnetic resonance imaging studies of the MCC have revealed conflicting results with regard to atrophy of this region in HD (Hobbs et al., 2011; Dogan et al., 2012). In turn, the lack of understanding of the pathological changes occurring in the global MCC warranted extensive study into this region to determine the extent to which the MCC is affected in HD. The MCC is of particular importance as the MCC confers a role in cognition, has connectivity to limbic areas, though less extensive than the ACC, and houses the cingulate motor areas, along with connections to motor-related regions of the brain. Therefore, potential pathology in the MCC can contribute to motor, mood, and cognitive symptoms that patients may present with.

A significantly greater huntingtin burden has been observed in the MCC of the HD brain when compared to the control brain confirming the presence of disease-related changes in this region. Interestingly, microglia have been shown to closely relate to huntingtin aggregates, showing a co-expression pattern synonymous with either the autonomous expression of huntingtin in microglia or the engulfing of huntingtin by microglia. This is particularly intriguing as mutant huntingtin may be a trigger for the activation of a microglial inflammatory response. In recently published work glial changes in the MCC in HD were studied using immunohistochemical techniques on fixed post-mortem human brain tissue. This study reported a greater proportion of microglia in morphologies consistent with reactivity in the HD MCC when compared with controls (Palpagama et al., 2023; Figure 1). However, a decrease in the proportion of ameboid microglia in HD when compared to controls suggests that the apparent reactivity of microglia may not be aiding in the clearance of mutant huntingtin. Interestingly, despite morphological changes, microglia in the MCC did not exhibit increased expression of the classical activation marker HLA-DP/DQ/DR. These findings may be suggestive of early shifts in microglia in the MCC towards classical activation and that only a subpopulation of microglia reach activation. Furthermore, these findings may be indicative that microglial cells in the MCC are activated to some degree but do not express HLA-DP/DQ/DR. Importantly, this latter possibility warrants a discussion of our understanding of “microglial activation” as a state. Historically, microglia were considered to exist in distinct states as either resting microglia or microglia polarized to an “activated state”. Activated states were thought to fall within a spectrum with either a phagocytic subtype that initiates and sustains immune responses termed an “M1” state, or an anti-inflammatory “M2” state. While classically the M1/M2 paradigm was utilized to summarize microglial gene and protein expression changes following a stimulus, recent literature proposes that the consideration of microglia to fall into distinct states is oversimplified (Palpagama et al., 2019). Indeed, the array of microglial molecular expression profiles in addition to varied morphologies of microglial cells provides evidence for the deviation from the hypothesis of distinct activation states along an M1/M2 axis (Palpagama et al., 2019). Therefore, the need to comprehensively analyze microglial cell populations based on gene and protein expression profiles to conclude the potential contribution of microglia populations to pathology in a region-specific manner becomes evident.

Figure 1.

Increased microglial activation in the midcingulate cortex in Huntington’s disease is correlated with symptom-specific alterations in microglia number and morphology and astrocyte glutamate transporter expression.

Glial changes observed with the presentation of mood symptomatology suggest glial dysfunction in the midcingulate cortex may contribute to mood-related symptomatology in Huntington’s disease. Created with BioRender.com.

Only a few studies have explored astrocytic changes in the cortex, with the literature suggesting regional cortical astrocytic changes in HD. Previous investigations into astrocytic changes have been carried out in the prefrontal cortex, temporal cortex, insular cortex, mediofrontal gyrus, and anterior cingulate cortex; however, prior to our study, no examination of the MCC has persisted. To this point, we examined alterations in the glutamate transporter EAAT2 expression, which is specifically expressed in astrocytic processes, and observed potential astrocytic dysfunction in this region (Figure 1). Immunohistochemical studies revealed significant decreases in EAAT2-positive astrocyte size, area coverage, and density in the MCC in HD when compared with controls. Interestingly a symptom-specific reduction in EAAT2 was observed in patients who presented with mood symptomatology when compared to patients who presented with mainly motor symptom profiles. This suggests that regional dysfunctional glutamate buffering by astrocytes may contribute to the development of certain symptom profiles of patients (Palpagama et al., 2023). While these results are intriguing, the lack of human cortical studies highlights a critical need to investigate astrocyte dysfunction in the cortex and how it may be altered in a symptom-specific way.

The interplay between neuronal and non-neuronal cells suggests that these cell types contribute to the pathophysiology of HD. As such, though not the focus of the current perspective, it would be remiss not to mention the growing body of literature on oligodendrocyte dysfunction in the progression of HD. Impairment in the maturation of oligodendrocytes, impaired myelination, and adaptive myelination, the dysregulation of oligodendrocyte lineage transcription factors and myelinogenic genes, and abnormal lipid metabolism in oligodendrocytes have all been hypothesized to contribute to the oligodendrocyte dysfunction observed in HD (Casella et al., 2020; Ferrari Bardile et al., 2023). Interestingly, the proliferation of oligodendrocyte precursor cells has been observed in HD mouse models and may potentially confer a compensatory mechanism for oligodendrocytic dysfunction (Casella et al., 2020; Ferrari Bardile et al., 2023). Widespread white matter volume loss is a pathological change observed in HD, and the literature suggests that altered organization of fibers along with decreased myelin sheath thickness may underlie altered neuronal activity, axonal degeneration, and reduced connectivity (Ferrari Bardile et al., 2023). The key contribution of oligodendrocytes to myelination suggests that dysfunction of these cells may underlie white matter degeneration in HD.

To conclude, the contribution of microglia and astrocytes to the pathology of HD continues to be a focus of research. The hypothesis of an underlying contribution of chronic inflammation, exacerbated by glial cells, in the formation of a neurotoxic environment may underpin the widespread neuronal cell death in the HD brain. Regional degeneration has been established to form the basis of the formation of specific symptomology in patients. Therefore, the exhaustive study of microglial and astrocytic gene and protein expression in a region-specific fashion will enhance our understanding of the effect of the glial response in pathogenesis and may produce new targets for future therapeutic intervention.