The brain consists of a variety of cell-types (including neuron and glia), all of which have to play their part to ensure proper functioning of the brain. Studies focusing on the development of neural cells and their function within neural circuits have provided great insights into functioning of the brain. However, once the neural and glial cells mature to adopt their bona fide functions, how their cellular identity, fate and function is maintained throughout an adult lifespan remain rather poorly understood. This is an important question because aberrations in cellular-form and function often leads to pathologies. Thus, insights into mechanisms that preserve the normal form and functions of adult brain cells would not only provide better understanding of neural cell-biology, but could also provide critical insights into varied neuro-pathologies. 

            Research in our lab seeks to understand mechanisms that maintain the homeostatic functioning of mature cells in the adult brain. We focus on three different cell-types in the brain: adult neural stem cells, neurons, and microglia. Each of these cells are specialized in their form and function that needs to be maintained throughout an adult lifespan, presumably through cell-specific gene-regulation. Using sophisticated mouse genetic-tools that allow tracking cells in live animals, complemented with behavioral, electrophysiological, molecular, morphological and single-cell transcriptomics and genomics studies, we investigate cells in the adult brain in the following three directions, under the overall theme of adult brain homeostasis and neuroinflammation.

A) Fate-restrictions and alternate potentials of adult neural stem cells (adult NSCs):

Similar to embryonic NSCs, the adult brain NSCs are multipotent, restricted to only producing neurons, astrocytes and oligodendrocytes. Mechanisms that govern this restrictive fate-potential of NSCs in the adult brain is crucial for maintaining normal adult neurogenesis. We recently demonstrated that active ‘fate-restriction’ mediated by Tcf4 is critical for maintaining adult NSC in its normal functional-state (Shariq, M. et. al, Science Advances, 2021). In absence of this restriction, the adult NSCs manifest a latent ‘inflammatory’ potential, that adversely effects adult neurogenesis and induces inflammation in the brain parenchyma. Our study provides the first ‘proof-of-principle’ evidence for an inflammatory potential in adult NSC, and identify a molecular regulator that continually keeps this alternate potential suppressed. 

Adult neural stem cells’ fate potential: The neural stem cells of the adult brain are also known as the Radial Glial-like cells or RGL because of their ancestral closeness to glia (astrocyte). While astrocytes are multipolar, RGL cells are polar with a single process extending radially and branching at top to give the specific morphology of RGL. RGLs can undergo asymmetric division, to give rise to a neuronal precursor or an astrocyte while self-renewing itself, or symmetric division which results only in self-renewal. Thus, the adult NSC/RGL are known to have potential for making CNS-friendly cells: neurons, astrocytes and RGL. However, we demonstrate that adult NSC has a latent myeloid-inflammatory potential that is prevented by continual expression of Tcf4. In absence of Tcf4, the adult NSCs transform into a myeloid-like cell-type secreting inflammatory factors that cause inflammation in the neurogenic niche; the dentate gyrus.

            Given that inflammation has a constructive role in some physiological circumstances, could this latent potential be evoked in adult NSC in specific contexts? Also, if this potential is indeed there in adult NSC, what would be its implications in the context of regenerative usage of the adult NSC? What would be its implications in contexts of stress and anxiety, where adult neurogenesis is thought to play a role. In similar vein, physiological contexts where parenchymal inflammation is observed, could there be a role of adult NSC? Could adult NSC transformation contribute to potential triggers to neuroinflammation in aging and in neurodegenerative diseases? We investigate the adult NSC to examine some of these questions. 

B) Mature neuronal maintenance: preserving structure and function of adult neurons 

A second major interest in the lab is to understand how adult neurons maintain their dendritic structure and function throughout a lifespan. The topic of adult neuronal maintenance is important because neurons are one of the longest-living cells in our body which are vulnerable to degeneration, yet have little capacity for turnover. An important aspect of neuronal function is its dependency on the physical connectivity within a neural circuit that is enabled by its dendritic arbour. These dendritic connections are laid down during embryonic and early-postnatal development, and need to be maintained throughout a lifespan. However, how and whether the dendritic structure of mature neurons are actively maintained through gene regulation remain elusive.

            Our recent study (Sarkar, D. et. al., Translational Psychiatry, 2021) has uncovered novel gene-networks that continually maintain the dendritic structure and membrane properties of adult neurons. Uncovering important insights about maintenance of form and function of adult neurons, this study revealed that even after their full maturation and functional connectivity within a circuit, adult neurons retain the ability to change their dendritic structure, and therefore their connections. Further, it provided evidence that structure and activity of adult neurons are under active gene-regulation, and revealed a genetic-network that continually works to subvert structural changes in adult neurons during the steady-state.             These revelations open up new directions of investigations into adult neuronal cell-biology. Given that adult neurons maintain an ability to change their dendritic tree, how and when do neurons use this? There are examples of dendritic structural changes in the adult brain, however the mechanisms and functional relevance of dendritic structure changes in adult neuron remain elusive. We seek to identify physiological contexts where dendritic changes could be useful for adult neurons, and probe potential molecular mechanisms that may guide and regulate this process for aiding in circuit functions.

Proactive maintenance of mature neurons in adult brain. Mature neurons in the adult brain have a particular structure and activity pattern, which allows for its proper function within a neural circuit. How the specific structure and membrane properties of a mature neuron are maintained throughout a lifespan remain poorly understood. We discovered that continual expression of the transcription factor Tcf4 is critical to both the structure and membrane properties of projection neurons in the adult brain. By ablating Tcf4 expression in excitatory neurons in the adult brain, we demonstrate that the Tcf4-deleted neurons rapidly morph their dendritic tree and fire more action potential due to profound changes in its membrane properties. Further examination of the transcriptome in Tcf4-deleted neurons revealed that Tcf4 suppresses a large network of genes, the majority of which are known to play a role in membrane and ciliary pathways. This study highlighted the inherent potential in mature adult neurons for significant dendritic structural changes, and a possible role of ciliary pathways in this process, thereby revealing possible new players in maintenance of mature neuronal structure and function.

            Another topic of interest in the ‘neuronal-maintenance’ theme is neuronal-heterogeneity. There are evidence suggesting that neuronal population, even within a specific sub-type, are heterogenous for their gene expression. However, the functional relevance of this diversity in the context of neuronal and circuit functions remain unknown. We investigate this by focusing on hippocampal pyramidal neurons in the adult brain.

C) Microglia homeostasis in the adult brain

A third area of investigation under the overall theme of brain homeostasis, is to gain insights into the maintenance of the brain-resident macrophage, the microglia. Compared to neurons and the macroglia (astrocytes and oligodendrocyte), the microglia in the central nervous system have a distinct ancestral origin, a wider repertoire of functions, and the unique capability to turnover in situ throughout an animal’s lifespan. Microglia in a healthy-adult brain is often described as the ‘resting microglia’, although it is anything but ‘resting’. The homeostatic microglia are known to perform myriad of functions that aid in neural activity. These functions range from formation and elimination of spines on neurons, trophic factor regulation for myelination to synaptic pruning. But, microglia are more popularly known as the immune cells of the brain. 

Varied manifestations of microglia: Microglia adopt different manifestations during homeostasis in a healthy adult brain and during immune activation. Shown are ‘homeostatic microglia’ in the adult brain with thin spread-out processes, also described as the ‘ramified-state’. In contrast, in the state of immune-activation, microglia adopt several different forms and functions, showing thickening and shortening of its processes, enlarged cell body and lysosomes, collectively termed as the ‘ameboid state’. The vastly different functions of microglia in a healthy brain versus under immune-activation depend on distinct gene-regulations. Whether these are dynamic manifestations of microglial-states that could be modulated through gene-regulation, or if there are distinct functional subsets that are amplified for specific functions remain an open question in the field.

              Microglia are the tissue-resident macrophages in the brain, capable of evoking the full repertoire of innate immune functions including phagocytosis, cytokine secretion, and antigen presentation. However, these immune functions are induced only under physiological circumstances of perturbation, such as during injury, infection and pathology. Interestingly, immune-activation of microglia has been noted in almost all neurological pathologies, be it developmental, degenerative or psychiatric. 

            During our investigations in microglia, we have recently uncovered an age-dependent sensitivity of microglia to Cre-mediated DNA toxicity and Type-1 Interferon signaling (Sahasrabuddhe, V et. al., Cell Reports, 2022), a revelation critical for genetic manipulation of microglia in vivo. Further to our interest in homeostatic regulations in adult brain cells, we seek to decipher gene-regulation that underlies the switch from an ‘immune-activated’ to ‘homeostatic-state’. This is an important question in the field because a prolonged state of immune-activation in microglia is thought to be detrimental for the brain, and is associated with many neuro-pathologies. Given that homeostatic microglia perform important functions in the healthy brain during the steady-state, microglia restoration to ‘homeostatic-state’ after an immune-response is critical for normal brain-functioning. However, how and if immune-activated microglia can be fully restored to its homeostatic-state remain unclear. To this end, we employ several different physiological models such as injury, infection and ablation-induced repopulation, to examine if common regulatory principles are applied in divergent conditions of immune activation, for regaining homeostatic microglia after an immune response.