Recombinant human stress protein Hsp70 as a perspective neuroprotective agent
Alzheimer’s disease (AD) is the most prevalent neurodegenerative pathology in the growing population of elderly humans and leads eventually to dementia and death. Despite tremendous efforts, no effective treatment for AD is currently available. The severity of cognitive impairment in patients with AD usually correlates with the extent of the observed abnormality of neurons, including the quantity of neurofibrillary tangles, the decrease in synaptic density, the enhanced concentration of soluble Aβ-amyloid oligomers, and the amount of neurons that die by apoptosis (1). Heat shock proteins (Hsps) have emerged as critical regulators of neurodegenerative processes associated with protein misfolding in the brains of AD patients (2). Various data suggest that Hsp70 and other molecular chaperones function as a complex neuroprotective system, which fails in the brains of AD patients. Hsp70 confer protection against oxidative stress and inflammation which play a major role in many age-related pathologies including Alzheimer disease (AD). Despite this, the direct therapeutic effect of exogenous Hsp70 administration on longevity in mammals has not been demonstrated.
To test possible neuroprotective effect of recombinant human Hsp70 we used two separate mouse models of AD-like pathology, bulbectomized NMRI mice and transgenic 5XFAD mice. Bulbectomy in 10-weeks aged mice leads to rapidly development of AD symptoms such as Aβ-peptide accumulation and loss of cortical and hippocampal neurons, and behavior disorders (3). 5XFAD mice co-express the Swedish (K670N/M671L), Florida (I716V) and London (V717I) mutations in human Aβ-PP(695), and M146L and L286V mutations in PS1, with expression of both transgenes driven by Thy1 promoter. This strain is characterized by rapidly neurodegeneration and Aβ plaques formation like in the case of human AD. Sham operated (without bulb aspiration) NMRI or untreated NMRI mice were used as animal control. All mice were treated with Hsp70 (2 ug/mouse in form of intranasal injections) daily for 21 days. Termally denatured Hsp70, physiological solution and bovine serum albumin were used as treatment control for the same time.
We have shown that recombinant Hsp70 expressed in eukaryotic cell cultures as well as in Escherichia coli rapidly enters specific brain regions after intranasal administration in mice. We confirmed localization of recombinant Hsp70 conjugated with Alexa Fluor 647 in mouse brains following intranasal injections (Figure 1). The experiments were performed in parallel with iodinated Hsp70 injections. Three hours after administration, fluorescently labeled Hsp70 was readily detected in the hippocampus and temporal cortex in the experimental but not in the control untreated mice (Figure 1). In most cases, the injected protein exhibited intracellular localization, which was concentrated in the perinuclear zone. Finally, intranasally injected fluorescent labeled recombinant Hsp70 observed in brain regions including specific areas (e.g., hippocampus) where neurodegeneration predominantly develops in AD patients and in the process of aging.
To resolve this issue, we decided to use 125I-labeled recombinant Hsp70. We demonstrated that the protein rapidly (within 30 min) enters different brain regions after administration but apparently undergoes rapid proteolysis and is not detectable in an intact form
(70 kD band) after one hour. Labeled BSA, which was used as a control, exhibited similar pattern of penetration and degradation in the brain. These results shown that Hsp70 effectively crosses the blood-brain barrier when administered intranasally.
In the next step we have shown that intranasally Hsp70 treatment reduces the accumulation of Aβ-peptide in the brains of OBX and 5XFAD mice. The total amount of soluble and insoluble fractions of Aβ in the untreated groups of OBX and 5XFAD mice was significantly higher than that in the control and groups subjected to Hsp70 treatment (p < 0.001) (Figure 2). Importantly, Hsp70 treatment per se did not affect the basal level of Aβ in the control groups. Thus, Hsp70 treatment effectively protects the brains of mice from the accumulation of potentially toxic Aβ-peptide in both animal models of AD.
It is known that 5XFAD mice develop characteristic Aβ plaques in 2 – 3 months after birth (3, 4). Intranasal Hsp70 administration strongly diminished the plaque density in the neocortex and CA1 region of the hippocampus in Hsp70-treated 5XFAD mice (Figure 2).
Despite the pronounced accumulation of Aβ, OBX mice do not develop plaques, but do demonstrate death of neurons in the brain (4). Therefore, we explored the effect of Hsp70 on neurons morphology and survival in the cortex and the hippocampus of OBX animals. The proportion of pathologic neurons decreased drastically in OBX+Hsp70 animals. The data demonstrate that the Hsp70 treatment prevents the development of pathology in neurons of the OBX group. Consistent with the protection of cells, Hsp70 significantly improved neuronal survival in OBX mice. Quantitative analysis revealed that the Hsp70-treated OBX mice had significantly more intact neurons in the temporal cortex and hippocampus. Therefore, Hsp70 treatment of OBX mice protects neurons from deterioration and death in brain areas most affected in AD patients.
Exogenous Hsp70 protects spatial memory decreased in OBX and 5XFAD mice in Morris water maze test. It is evident that untreated OBX and 5XFAD mice had severely impaired spatial memory. In contrast, OBX and 5XFAD mice treated by Hsp70 displayed a significant preference for the target sector. We therefore conclude that subchronic intranasal injection of Hsp70 protected spatial memory in both OBX and 5XFAD animals.
In conclusion, we can summarize that the intranasal administration of recombinant human Hsp70 drastically alleviates all symptoms, including memory loss, neuronal death, cellular aberrations, and accumulation of the Aβ-peptide in both AD-models explored.
In our experiments we also examined whether the intranasal administration of exogenous recombinant human Hsp70 can promote longevity in male and female mice. Interestingly, long-term (chronic) introduction of Hsp70 did not affect the longevity of females significantly, while increased the lifespan of males (Figure 3). The Hsp70 treatment also normalized the synthesis of synaptophysin and decreased accumulation of lipofuscin which represent the marker of aging and neurodegeneration processes (Figure 4).
We hypothesized that Hsp70 exerts its protective effects on behavior by decreasing the production and/or accumulation of damaged proteins in the brain. A majority of intracellular proteins are degraded by proteasomes; thus, the catalytic activity of proteasomes is critical to the homeostatic process. Because proteasome activity decreases with age in brain (5), we investigated whether eHsp70 treatment altered proteasome activity in the brains of treated mice. Caspase-like activity in the cortices of control animals was comparable to that detected in brains of Hsp70-treated mice. In contrast, chymotrypsin-like activity in mice of the Hsp70-treated group was significantly increased. We analyzed the subunit composition of proteasomes after intranasal Hsp70 injections and observed a significant elevation in the Rpt6, β6 and β5i subunits expression. These findings suggest that the elevated chymotrypsin-like activity in the brains from eHsp70-treated animals is likely due to the increased amount of proteasomes containing the β5i subunit.
In parallel with effects on biochemical level Hsp70 treatment leads to increasing of neuronal density and repairs spatial memory loss in aging mice as well as in AD models (OBX and 5XFAD).
Taken together, our findings establish exogenous human Hsp70 as a practical pharmacological agent for the treatment of various neurodegenerative diseases (as well as the consequences of brain injury) and possibly aging.
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Figure 1. Distribution and stability of recombinant Hsp70 after penetration into the brain of NMRI mice. (А – olfactory bulbs, B – n. raphe dorsalis, С, D – hippocampus, Е – cerebellum). Localization of the label in the perinuclear region is indicated by white arrows. F – control mice (injection of unlabeled Hsp70).
Figure 2. A, B – the level of β-amyloid in the brain of mice: a sham-operated (SO), bulbectomized (OBX) NMRI strain and transgenic 5XFAD, control and subjected to intranasal injections of recombinant Hsp70 (+Hsp70). C – the density of amyloid plaques in 5XFAD mice without Hsp70 treatment and after Hsp70 injections. Micrograph of a slice of NMRI mouse brain is given as the example of a negative control (without the formation of β-amyloid plaques). ## p ≤ 0.01; ### p ≤ 0.001.
Figure 3. Effect of chronic eHsp70 treatment on longevity. (A) Longevity of old animals when Hsp70 treatment was started at 17 months of age and lasted until animals’ death. (B) Longevity of middle-aged animals when eHsp70 treatment was started at 12 months of age and lasted until animals’ death.
Figure 4. Effect of eHsp70 administration for 5 mo on molecular markers of aging. Levels of synaptophysin (A) or lipofuscin (B) were measured in the brains of Hsp70-treated and control old mice. *** p ≤ 0.01.