Recent in-vitro data demonstrated that many types of cells (ranging from cardiomyocytes
and fibroblasts to algae and yeasts) experience a circadian oscillation of protein home-
ostasis. This process is mTOR-regulated and influences cytosolic macromolecular crowd-
ing; as the protein degradation phase is associated with an increased condensation state.
The changes in the aggregation state of cytosolic proteins are associated with a variation of the intracellular os-
motic pressure, which is counterbalanced by an electro-neutral active ionic transport that
modifies intracellular ionic concentrations N a+, K+ and Cl− to maintain the osmotic
homeostasis and cell volume. Hence, ionic concentrations and molecular crowding show
antiphase-correlated oscillations and are circadianly regulated, as impairment of the circa-
dian clock causes a progressive attenuation of the oscillation of mTOR activity and ionic
concentrations.
To date, there is evidence these oscillations modulate cardiomyocyte electrophysiology
and it could be possible that this phenomenon occurs also in cortical neurons, influenc-
ing neuronal and circuital functions. Supporting this hypothesis there are observations
of a diurnal variation of intracellular chloride concentration [Cl−]in that alters cortical
excitability and susceptibility to seizures (still unpublished data).
The aim of this thesis is to provide some initial data to support the circadianity of the
compensation between protein and ionic homeostasis in cortical neurons, as preliminary
immunohistochemistry data against phosphorylated ribosomal protein S6 (pS6), which
is an mTOR substrate, on cortical neurons, demonstrated a strong circadian oscillation
of mTOR activity. Furthermore, to formally demonstrate the interconnection between
protein homeostasis and neuronal excitability, we are studying a mouse model of human
lipofuscinosis, associated with epileptic phenotype, the CLN1 KO. In this model, the
autophagic process is impaired, leading to aberrant protein accumulation and loss of cir-
cadian proteostasis oscillation that could mediate, through ionic concentration imbalance,
the altered neuronal excitability.