Redox modulation of cellular stress response and lipoxin A4 expression by Hericium Erinaceus in rat brain: relevance to Alzheimer’s disease pathogenesis

AD has gained widespread attention because of its high economic costs, which have
reached $400 billion per year in the USA alone, as well as the social costs, which
are more difficult to quantify 26]. AD-related pathological markers include a progressive death of neurons in specific
areas with an accumulation of intracellular neurofibrillary tangles (NFTs) and extracellular
depositions of amyloid plaques (APs). NFTs are composed of the misfolded hyperphosphorylated
microtubule-associated protein Tau (MAPT or Tau), whereas APs are extracellular deposits
of misfolded and aggregated amyloid-beta peptides (A?) 27], 28]. Because both NFTs and APs are persistently found in areas with severe neuronal death,
these proteins were considered to be the main cause of neuronal loss and the emergence
of dementia, which is a crucial symptom of AD; however, numerous drug trials based
on these proteins have failed to provide a useful AD therapy 28]. A post-mortem study demonstrated that the misfolded protein accumulation is a shared
pattern in many neurodegenerative diseases, including AD 25], concurring to the conclusion that accumulation of misfolded proteins is a prominent
potential cause of neurodegeneration in AD 29]. Recently, the involvement of neuroinflammation and microglial activation in the
pathogenesis of AD has been emphasized by compelling evidence from basic and clinical
research studies indicating that inflammation induced by A? is intimately associated
with the development of AD neuropathology 19]. Relevant to the central role of neuroinflammation in AD pathgenesis, are recent
advances in knowledge of the mechanisms of inflammatory resolution, identifying lipoxins
as attractive therapeutic tools to treat diseases in which inflammation is involved
22]. LXA4 is generated via the lipoxygenase pathway during cell-cell interactions in
inflammatory conditions, whereas aspirin-triggered LXA4 (ATL), a molecule that displays
the same anti-inflammatory activities as the native lipoxins, is generated after the
acetylation of cyclooxygenase-2 and is more resistant to metabolic inactivation 23]. Lipoxins potentiate inflammatory resolution by means of potent agonistic actions
at the G-protein coupled receptor, termed LXA4 receptor (ALX/FPR2). Activation of
ALX by LXA4 reduces many endogenous processes, such as neutrophil and eosinophil recruitment
and activation, leukocyte migration, NF-kB translocation, and chemokine and cytokine
production 22]. Likewise, evidence shows that LXA4 signaling primes macrophages for chemotaxis and
enhances phagocytosis of microorganisms and apoptotic cells. In the nervous system,
LXA4 protects neurons against experimental stroke and A?
₄₂
toxicity by modulating inflammation. In addition, lipoxins inhibit inflammatory pain
processing through their actions on astrocytic activation in the spinal cord 30], 31]. However, the ability of LXA4 signaling to modulate neuroinflammation and AD pathology
in vivo has not been yet completely elucidated.

Mushrooms provide a great potential as a polypharmaceutic drug because of the complexity
of their chemical contents and different varieties of bioactivities. If available
evidence suggests anti-oxidants, anti-tumor, antivirus, anti-cancer, anti-inflammatory,
immune modulating, anti-microbial, and anti-diabetic activities from mushrooms 32], however, contrarily to plant herbal medicines, which are widely explored and relatively
more advanced, the brain and cognition health effects of mushrooms are in the early
stages of research. Here, by extending previous finding on nutritional approaches
to neuroinflammation, we provide experimental evidence that administration of H. erinaceus for 3 month to rats results in up regulation of vitagenes, in particular Hsp70, HO-1
and Trx, an effect associated with increased synthesis of LXA4 in different brain
regions of rat. This latter, an endogenous eicosanoid, is emerging as an important
resolvin, a class of compounds endowed with the capability to promote resolution of
inflammation, therefore suggesting that nutritional modulation with H. erinaceus, through redox-dependent vitagene network might activate endogenous “braking signal”
processes impacting the inflammatory process. We also provide evidence of neuroprotective
action of H. erinaceus when administered orally to rat. Expression of LXA4, measured in different brain
regions after oral administration of a biomass H. erinaceus preparation for 3 month increased significantly in all brain regions examined, as
compared to control group of animals, particularly in cortex and cerebellum, followed
by substantia Nigra, striatum and cerebellum. LXA4 up-regulation was associated with
an increased content of redox sensitive proteins involved in cellular stress response,
such as Hsp72, HO-1 and Trx. We show that SN exhibited lower LXA4 content respect
to other brain regions examined, both in control and mushroom stimulated animals.
This finding is relevant to AD and PD pathogenesis, particularly to theories connecting
aging and neuronal degeneration with oxidative damage. SN neurons are depleted during
physiological aging and even more so in all neurodegenerative processes associated
with Parkinsonian symptoms. 33]–37]. In addition, we demonstrate that H. erinaceus treatment resulted in a significant increase of LXA4 in most of the brain regions
examined and modulated expression of cytoprotective proteins, such as HO-1, Hsp70
and Trx. Our results are consistent with recent evidence obtained in mice, showing
neuroprotection by H. erinaceus on Ab25–35 peptide-induced cognitive dysfunction 38], 39]. In this study the powder of H. erinaceus was mixed with a normal powdered diet and the Ab25–35 peptide was administered by
intracerebroventricular injection. The results revealed that H. erinaceus prevented
impairments of spatial short-term and visual recognition memory induced by Ab25–35
in mice. Furthermore, human trials with H. erinaceum derivatives also have showed promising results in patients with dementia based on
Revised Hasegawa Dementia Scale (HDS-R) 40].

Our results indicating that nutritional modulation of critical proteins involved in
brain stress tolerance can be achieved via supplementation with a well characterized
strain of H. erinaceus are relevant to those theories connecting proteome control quality failure with age-associated
neurodegenerative diseases. Consistent to this notion, in AD pathology, the accumulation
of APs composed of A? aggregates and neurofibrillary tangles NFTs composed of misfolded
Tau proteins, accumulation of these proteins as consequence of faulty protein quality
control mechanisms, is associated with a deficit in those mechanisms participating
to induction of cytoprotective proteins (Hsps) or, more in general, involved in the
cellular pathways of stress tolerance. It is conceivable that in these conditions
administration of H. erinaceus mushroom, which increases the redox potential associated with induction of vitagenes,
may help vulnerable neurons to resist to proteotoxic insults and hence to apoptotic
neurodegeneration. This is furtherly corroborated by the finding indicating that restoration
of normal proteostasis is crucial for neuronal survival 41].

The molecular chaperone Hsp70 protects cells from injury by binding damaged proteins
under stressful situations. Members of the 70 kDa-heat shock protein family (Hsp70s)
are, in their function as molecular chaperones, involved in folding of newly synthesized
proteins and refolding of damaged or misfolded proteins, as well as in assembly and
disassembly of protein complexes. All human Hsp70s have highly conserved domain structures
42]. They consist of an N-terminal ATPase domain, a middle region and an N-terminal peptide
binding domain. However, they differ in expression patterns, cellular localization
and function. There are Hsp70’s specifically located in the endoplasmatic reticulum
(Grp78, also known as BiP) and in the mitochondria (Grp75, also known as mortalin).
However, the members which are mainly located in the cytosol and nucleus are the heat
shock cognate protein 70 (Hsc70) and the heat shock protein 70 (Hsp70). Cellular stress
often leads to protein unfolding and, therefore, to increased protein hydrophobicity,
which may result in the formation of toxic protein aggregates 42]. As recently demonstrated, Hsp70 expression is induced under the mild oxidative stress
conditions, when oxidative damage to proteins leads to their unfolding 43], and the heat shock response is activated driving increases in the expression of
molecular chaperones, which reaches about two-fold the baseline levels 43]. Although HSP’s can refold mildly disordered proteins, it is clear that HSP’s are
not able to repair covalently-modified oxidized proteins or to reverse oxidative protein
modifications, which results in increased protein hydrophobicity, as triggering signal
for the activation of a highly regulated and rapid series of events, called the ‘heat
shock response’ (HSR). Heat shock transcription factor 1 (HSF1) is bound to a complex
of heat shock proteins (Hsps), such as Hsp70 and Hsp90, during non-stressed conditions
and, therefore, kept in an inactive state. When Hsps recognize hydrophobic patches
of damaged and unfolded proteins, the Hsps dissolve from the complex with HSF1 in
response to cellular stress. This event is followed by HSF1 trimer formation, which
further leads to the activation and translocation of the transcription factor into
the nucleus, where the trimer binds to the heat shock gene promoters, the so called
heat shock elements (HSEs). This leads to the fast expression of Hsps 44]. Moreover, the heat shock genes do not contain introns, which further accelerates
their expression. An unfolded protein that binds to Hsp70 may be either refolded into
its native non-toxic conformation and then released, or may stay bound by Hsp70 to
protect non-damaged molecules. Since most oxidative protein modifications are not
repairable due to their covalent nature, the majority of oxidized proteins are degraded
by the proteasomal system.