"Feeling the Distance: Investigating the molecular mechanisms of intrinsic cell size sensing in neurons using stem cells, bioengineering and imaging"

Neurons are specialised cells that connect to each other and to other cells types across our bodies and rely information and commands from our nervous system to the other organs. Like any other cell, their shape is intimately linked to their function, and as some of the target organs can be sometimes at considerable distance from the brain, neurons themselves have to span vast distances.

For example, spinal Motor Neurons need to connect the spinal cord with every single muscle fibre in our body and can sometime have axons -the long protrusions that neurons use a "cable" to connect to other cells" exceeding 1m in length. This extreme shape poses a challenge for the neurons: the main centre of production for resources (proteins and RNA) is in the cell bodies, but the main site of high demand and consumption is at the end of a 1m long axon, with the two sites connected by a chain of transporter molecules. Moreover, demands in the synapses at the end of the axon need to be matched by production of component in the soma, but for very long axons demands might change swiftly and the vast distance separating the end of the cell make it impossible to run a "just-in-time" production line.

For these reasons, neurons have adopted mechanisms to create local production and regulation of resources away from the cell body and into the axon, rendering the latter more independent and capable of buffering fast changes in demand.

One key question in all this, is how do neurons sense their own length and decide to enact these length-dependent adaptation, and whether there is one specific class of signals responsible for sensing axonal length or if the process is more guided by a complex interplay between supply and demand across multiple fronts.

This is a fundamental question for basic neurobiology, but it is also very relevant for understanding the basis of several human diseases, as in several neurodegenerative disorders imbalances in supply and demand of energy at the far end of the axon seems to be some of the earlier observable events in the chain of problems that ends with the death of certain neurons, like in Amyotrophic Lateral Sclerosis.

Up until now it has been complex to study this mechanism systematically, as in animal models it is not possible to systematically change the length of axons in a simple way, and cell culture systems generally have very short neurons. As a result, most of the proposed mechanisms for this sensing capacity of neurons is centred around relatively short axons, usually well below 1mm.

We have developed a novel platform that combines bioengineering, human stem cells and advanced imaging to create ordered arrays of human motor neurons with controllable length up to and exceeding 1cm, which we have used to successfully demonstrate that several important mechanisms are fundamentally altered in the axons when a certain length is reached (i.e. "threshold length") and we therefore perfectly poised to systematically study the mechanisms behind neuronal size sensing, to understand what determines this "threshold length".

To do so, we propose to use our platform and systematically alter all the different pathways we observed changing with the axonal length, to determine if any of them is directly responsible for determining the "threshold" length, and if so what is the sequence of events that leads the neurons to enact these adaptations. Our hypothesis is that it will be the dynamics of ATP (the cell's unit of currency for energy and basis of all other function) that will be one of the early -if not the first- feedback system, which determines at which length energy levels are no longer sustainable and more local processes for production and upkeep need to be implemented.

The main goals of this project are to understand in detail how human neurons sense their length and adapt to maintain their function across large distances and to determine what are the mechanisms involved in this length sensing feedback.

Neurons have extremely polarised architecture, with axons sometimes spanning over one meter in length separating synapses from cell bodies. To maintain their function across such vast distances neurons enact specific adaptive mechanisms to locally regulate homeostasis and metabolism.

One fundamental question still not completely understood is how these adaptations are triggered and what is the mechanisms that leads to these switches from centralised to delocalised processes. Some mechanisms for this feedback sensing process have been proposed, but their mainly apply to shorter neurons (generally below 1mm), and have generally studied in murine models.

We have developed a bioengineered, human induced pluripotent stem cell (iPSC) based platform to reliably generate arrays of human spinal motor neurons with highly elongated axons (>1cm) in a controlled fashion (LAM arrays). With this platform we have demonstrated quantifiable changes in mitochondria, local synthesis and other pathways involved in axonal homeostasis, when comparing long neurons to shorter ones, with the behaviour switch occurring at specific distances from the soma, which we termed "threshold length".

We propose to use our platform to perform a series of mechanistic studies to systematically alter the main pathways involved in these length dependent adaptations and understand the molecule series of events that determines the observed "threshold length".
By altering each pathway separately (mitochondrial dynamics, local protein synthesis, ATP and Ca2+ dynamics) we will be able to determine and which ones serve as primary feedback signalling for the neuron and what is their causal relationship.