Carnosine is an endogenous dipeptide highly expressed throughout the brain that has been suggested as a therapeutic tool in the treatment of AD, because the compound can act as an endogenous anti-oxidant, free radical- and metal ion chelator, and also has neuroprotective activity against in vitro Aβ-induced toxicity 
. Thus, the major aim of this study was to evaluate the effect of dietary carnosine supplementation in a model of AD that develops an age-related neurodegenerative phenotype that is driven by intraneuronal deposition of Aβ and accumulation of h-tau 
. We chose to treat only male 3xTg-AD mice as female hormones are known to negatively influence the activity of Zn2+
and differently affect the disease progression 
, therefore producing confounding effects. We chose to use PS1-KI animals as control group as these mice overexpress mutant Presenilin-1 gene (M146V substitution) but, by lacking the expression of mutant APP and h-tau, do not show Aβ or tau-dependent pathology nor AD-related cognitive deficits 
Carnosine has been shown to protect against Zn2+-mediated toxicity in cell cultures and that activity has been linked to the chelating properties of the compound. The peptide has indeed been shown to complex Zn2+ in acqueous solution but, to date, there were no experimental data demonstrating its chelating capability in biological systems. We tested this hypothesis in cultured glial cells and show that the peptide is in fact able to chelate [Zn2+]i ().
In our study, we also found a very potent effect of carnosine in rescuing mitochondrial dysfunctions in aged 3xTg-AD mice. As discussed above, mitochondrial deficits are emerging as key players in AD  
and, in line with observations indicating that Aβ and tau synergistically impair OXPHOS complexes 
, we found signs of potent deregulation of mitochondrial respiration in our AD mice. In 3xTg-AD mice at 12–14 m.o.a., we observed a reduction in the activity of complex I, II and IV in the hippocampus as well as of complex I and IV in the cortex. Interestingly, carnosine supplementation not only prevented such deficits but, in the hippocampus, we found that complexes II and IV activity of carnosine-fed AD mice actually increased over baseline (). Such results can be linked to the antioxidant activity of the dipeptide, a property that can prevent ROS-dependent mobilization of [Zn2+
. Such activity can block Zn2+
-dependent mitochondrial dysfunction 
, and inhibit the overproduction of nitric oxide 
, a process strongly potentiated by Zn2+
that eventually contributes to a self perpetuating mobilization of the cation. In addition, carnosine may have a direct effect on Aβ deposition and mitochondrial function by acting as an osmolyte as shown in the case of its action on methylene blue and cytochrome c oxidase 
When we analyzed the effects of carnosine on amyloid and tau pathology we found that the peptide is very effective in decreasing intraneuronal Aβ deposition in the hippocampus but does not affect the development of tau pathology.
Analysis of the effect of carnosine supplementation on cognitive deficits of 3xTg-AD mice showed a positive trend, indicating that it might have a beneficial role in preventing long-term memory deficits, although, this effect did not reach statistical significance. The sub-maximal effect we observed could be related to the fact that carnosine is able to greatly inhibit the Aβ load but not the appearance of tau pathology and these two molecular components are definitely acting synergistically in the development of the cognitive decline 
In the last few years a growing body of evidence is supporting the intriguing hypothesis that the alteration in the equilibrium of brain Zn2+
levels can be a significant contributing factor for AD 
. Interestingly, both excess as well as deficit of brain Zn2+
can favour AD-like pathology in AD animal models 
suggesting the existence of a finely tuned Zn2+
set point. Such hypothesis has been substantiated by a recent study indicating that deficits of synaptic Zn2+
promotes AD-like cognitive impairment by negatively interfering with glutamatergic and BDNF signalling 
. Thus, in such scenario, a decreased Zn2+
bioavailability induced by carnosine supplementation may in part exert negative effects on the neurotransmission and neurotrophic signalling that modulate cognitive functions, and in doing so, counteracts the positive activity on amyloid deposition and mitochondrial functioning. In that respect, it could be interesting to verify the possibility that more effective synergistic activity can be achieved when carnosine and Zn2+
are administered together in a combined form similarly to what has been described for the compound named Polaprezinc, a preparation that combines Zn2+
and L-carnosine 
. In theory, this compound, given the different Kd
of carnosine and Aβ can act as an homeostatic molecule that sequesters Zn2+
from Aβ but then releases a sufficient amount of the cation in the synaptic cleft to exert neurotrophic actions.
Another possibility that could explain the subthreshold effect on cognition is associated with changes in carnosinase activity as the enzyme has been found to undergo an age-dependent enhanced activity in brains of aging individuals and AD patients 
. Finally, it is also possible that a more robust effect could be revealed by extending these behavioural studies to a larger cohort of animals.
In summary, carnosine has a strong effect in restoring mitochondrial functioning and in counteracting amyloid pathology but these activities do not translate in a robust effect on cognition. These results suggest that, at least in complex AD animal models, addressing mitochondrial dysfunction and Aβ aggregation without a parallel intervention on h-tau deposition is not sufficient to promote major beneficial cognitive effects. Supporting this idea, recent reports have in fact indicated that therapeutic measures addressing Aβ overloads but unable to reduce the development of tau pathology do not prevent the development of cognitive deficits in 3xTg AD mice