Understanding the intricate process through which diverse brain cells manifest from their unique gene expressions offers more than just insights into normal brain functioning. It’s a key to comprehending the underpinnings of neurological disorders. In a groundbreaking study by MIT, the correlation between “molecular logic” in two specific neuron types of the Drosophila fruit fly and the subsequent divergence in their functions has been meticulously deciphered.
Decoding Cellular Diversity
Within the pages of Neuron, a consortium of neurobiologists hailing from The Picower Institute for Learning and Memory at MIT disclosed the divergent genetic landscapes of two closely related neuron subtypes within the Drosophila fruit fly. This variance extended to over 800 genes, constituting approximately 5% of the entire fly genome’s gene pool. Strategic manipulation of these differentially expressed genes allowed scientists to establish a clear link between gene expression and the apparent distinctions in cellular behavior.
Prof. Troy Littleton, the senior author of the study and the Menicon Professor of Neuroscience in MIT’s Departments of Biology and Brain and Cognitive Sciences, elaborated, “Amid the global endeavor in neuroscience to identify the myriad neuron types and outline their unique characteristics and gene expression profiles, this invaluable toolkit can pave the way for the exploration of how newly discovered disease-related genes interplay with specific neurons, shedding light on the most susceptible candidates in distinct brain disorders.”
The study focused on two neuron types emerging from the fly’s analog of a spinal cord, regulating muscle functions by releasing the neurotransmitter glutamate through synapses. Divergence between “phasic” and “tonic” neurons lies in their disparate mechanisms. Phasic neurons, connecting with numerous muscles, emit intermittent robust bursts of glutamate. Conversely, tonic neurons link to a single muscle, providing a steady, constant release of the neurotransmitter. This functional duality, echoing in human neurons, facilitates versatile control.
In the pursuit of delineating these disparities, Picower Institute postdoc Suresh Jetti spearheaded extensive analysis within Littleton’s lab. A profound comprehension of structural and functional distinctions between the cell types was coupled with a meticulous scrutiny of gene expression profiles, i.e., transcriptomes.
Upon closer examination, the two neuron types unveiled pivotal differences. Phasic neurons exhibited fewer synapses on each muscle, yet due to their widespread reach, they had to establish roughly four times as many synapses in total compared to tonic neurons. The latter displayed a greater number of inputs from other neurons, facilitated by more extensive dendrites. On the functional end, phasic neurons exhibited more potent signals upon stimulation and a higher propensity for transmission than tonic neurons. Notably, active zones (AZs), sites initiating glutamate release, indicated a greater influx of calcium ions in phasic neurons compared to tonic ones.
A novel revelation emerged concerning the distinct shapes of AZs in the two neuron types. While tonic AZs assumed a rounded configuration, resembling donuts, phasic AZs adopted triangular or star-like forms. Littleton speculated that this morphological variation potentially facilitated a higher concentration of calcium ions in phasic active zones, possibly explaining their augmented bursts of glutamate release in comparison to tonic neurons.
Unveiling Genetic Disparities
Gene expression assessment entailed Jetti’s utilization of the “isoform patchseq” technique. This demanding process involved the extraction of RNA from the nuclei and cell bodies of precisely identified tonic and phasic neurons within numerous flies. This exhaustive approach not only shed light on differential gene expression but also elucidated disparities in gene splicing and RNA editing.
Altogether, the study identified significant differential expression of 822 genes between the two neuron types. Notably, around 35 genes were associated with guiding axon branch growth, a crucial aspect underlying why phasic neurons innervate multiple muscles while tonic neurons target only one. Other differentially expressed genes were linked to synaptic structure and function. Additionally, over 20 genes hinted at variations in neuromodulatory chemicals to which each neuron type exhibited sensitivity.
The research illuminated the elevated prominence of transport proteins in phasic neurons, potentially accounting for their capability to manage the elevated demand for synapse formation across numerous muscles. Furthermore, while tonic neurons expressed “sialylation” genes, facilitating sugar attachment to synaptic membrane proteins, phasic neurons featured distinctive “ubiquitin” genes that facilitated protein breakdown.
Delving deeper, the team ventured into assessing the practical implications of these genetic differences. Perturbing specific ubiquitination genes led to excessive synapse growth in phasic neurons. Similarly, disruption of sialylation led to diminished synaptic growth in tonic neurons. Interestingly, tonic neurons displayed a fortyfold higher expression of the gene “Wnt4.” Interfering with this gene resulted in reduced synaptic growth in this subset of neurons.
Unraveling the Functional Nexus
A remarkable discovery centered on the heightened expression of a calcium-ion buffering gene in phasic neurons, exceeding that of tonic neurons by over thirtyfold. Upon mutating this gene to impede its function, phasic neurons, conventionally characterized by lower baseline calcium levels, exhibited elevated resting calcium levels akin to tonic neurons.
Moreover, a distinctive experiment displayed the distinct influence of specific cytoskeletal genes on the shape of active zones in each cell type. Downregulating a gene overexpressed in phasic neurons elongated their active zones, leaving those of tonic neurons unaffected. Similarly, downregulation of a gene highly expressed in phasic neurons induced non-round configurations in their active zones, again without impacting tonic neuron active zones.
In sum, the meticulous analysis laid the foundation for a rudimentary model elucidating the molecular basis of the divergent properties in the two neuron types. However, as Littleton emphasized, this is only the initial stride in unraveling the entirety of gene expression discrepancies that underpin the unique attributes of these neuronal subtypes.
Alongside Prof. Littleton and Jetti, the paper features co-authors Andres Crane, Yulia Akbergenova, Nicole Aponte-Santiago, Karen Cunningham, and Charles Whittaker.
Funding for this research was generously provided by The JPB Foundation, The Picower Institute for Learning and Memory, and the National Institutes of Health.
- MIT News – “Decoding Cellular Diversity in Neurons” URL: https://news.mit.edu/2023/decoding-cellular-diversity-neurons-0301
- The Picower Institute for Learning and Memory at MIT URL: https://picower.mit.edu/
- National Institutes of Health (NIH) – Neuroscience Research URL: https://www.nih.gov/research-training/medical-research-initiatives/neuroscience