HspB5 Activates a Neuroprotective Glial Cell Response in Experimental Tauopathy

Progressive neuronal death during tauopathies is associated with aggregation of modified, truncated or mutant forms of tau protein. Such aggregates are neurotoxic, promote spreading of tau aggregation, and trigger release of pro-inflammatory factors by glial cells. Counteracting such pathogenic effects of tau by simultaneously inhibiting protein aggregation as well as pro-inflammatory glial cell responses would be of significant therapeutic interest. Here, we examined the use of the small heat-shock protein HspB5 for this purpose. As a molecular chaperone, HspB5 counteracts aggregation of a wide range of abnormal proteins. As a TLR2 agonist, it selectively activates protective responses by CD14-expressing myeloid cells including microglia. We show that intracerebral infusion of HspB5 in transgenic mice with selective neuronal expression of mutant human P301S tau has significant neuroprotective effects in the superficial, frontal cortical layers. Underlying these effects at least in part, HspB5 induces several potent neuroprotective mediators in both astrocytes and microglia including neurotrophic factors and increased potential for removal of glutamate. Together, these findings highlight the potentially broad therapeutic potential of HspB5 in neurodegenerative proteinopathies

Toll-Like Receptors 2 and 3 Agonists Differentially Affect Oligodendrocyte Survival, Differentiation, and Myelin Membrane Formation

Toll-like receptors (TLRs) play a key role in controlling innate immune responses to a wide variety of pathogen-associated molecules as well as endogenous signals. In addition, TLR expression within nonimmune cells has been recognized as as modulator of cell behavior. In this study we have addressed the question of whether functional TLRs are expressed on oligodendrocytes, the myelinating cells of the central nervous system. Primary cultures of rat oligodendrocytes at different maturation stages were found to express TLR2 and, to lesser extent, TLR3. Immunocytochemical analysis revealed that both TLRs were localized at the cell body and primary processes and were excluded from myelin-like membranes. Interestingly, innate immune receptor ligands were able to modulate oligodendrocyte survival, differentiation, and myelin-like membrane formation, indicating that TLRs on oligodendrocytes are functional. In highly purified oligodendrocytes cultures, the TLR2 agonist zymosan promoted survival, differentiation, and myelin-like membrane formation, whereas poly-I:C, a TLR3 ligand, was a potent inducer of apoptosis. Together, these data indicate that, in addition to other neural cell types, also oligodendrocytes express functional TLRs, which play a role in regulating various aspects of oligodendrocyte behavior. (C) 2011 Wiley Periodicals, Inc

Phase IIa study profile.

<p>Phase IIa study profile.</p

Selection of therapeutic intravenous doses of HspB5 in humans.

<p>Therapeutic application of HspB5 in humans relies on activation of M2-like protective microglia and macrophage responses, while avoiding pathogenic IFN-γresponses by memory T cells. In Fig 2A, the existence of a substantial HspB5-reactive memory T-cell repertoire in humans is illustrated by a representative fresh PBMC sample from a healthy subject. Proliferative responses to HspB5 of CD45RO⁺ memory T cells in this sample are reflected by proliferation-induced dilution of the fluorescent tracer CFSE over 9 days in culture. With CFSE^dim CD45RO⁺ T cells representing proliferated memory T cells, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143366#pone.0143366.g001" target="_blank">Fig 1A</a> illustrates that a substantial part of all human memory T cells, an estimated one in about every 4,000 cells, do indeed respond to HspB5. In Fig 2B, it is illustrated, again with a representative example, that human T cells are triggered by HspB5 to release IFN-γ, but only when the HspB5 concentration exceeds a threshold of about 20 μg/mL. The protective macrophage response, on the other hand, is already activated by markedly lower μg/mL-concentrations of HspB5, as exemplified by IL-10 secretion as marker for protective responses. At these low sub-immunogenic concentrations, HspB5 can therefore be therapeutically exploited in humans. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143366#pone.0143366.g003" target="_blank">Fig 3C</a>, it is shown that single intravenous doses of up to 37.5 mg HspB5 leads to such sub-immunogenic peak serum concentrations in humans, remaining well below the 20-μg/mL threshold. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143366#pone.0143366.g003" target="_blank">Fig 3C</a> shows the mean ± standard deviation peak serum concentrations of HspB5 found in groups of 8 healthy subjects 10–20 min after receiving varying amounts of intravenous HspB5 during the Phase I study.</p

Effects of intravenous HspB5 on peripheral T-cell reactivity to HspB5 itself, and tetanus toxoid.

<p>Fig 3 illustrates the proliferative response <i>in vitro</i> of CD4⁺ (A) as well as CD45RO⁺ memory T-cells (B) to either 50 μg/mL HspB5 or 0.2 μg/mL tetanus toxoid as a control antigen, at various time points after a single intravenous dose of 12.5 mg HspB5 in 8 healthy subjects, as evaluated using a CFSE assay during Phase I. Percentages of proliferated CFSE^dim cells are expressed relative to all other lymphocytes detected by flowcytometry in the sample after 9 days in culture, including CD4/CD45RO-negative cells. The results not only confirm that following an intravenous dose of 12.5 mg HspB5, its levels remain at sub-immunogenic levels, but even illustrate significant suppressive effect on <i>in vitro</i> T-cell reactivity that is markedly more lasting for HspB5-reactive T cells than for tetanus toxoid-reactive T cells. Background proliferation found in cultures without any antigen was well below 1% in all cases. *: p< 0.05; ***: p<0.005.</p

Treatment-emergent adverse events occurring during the Phase I study in at least two different subjects following a single dose of HspB5.

<p>Treatment-emergent adverse events occurring during the Phase I study in at least two different subjects following a single dose of HspB5.</p

Decline in MRI lesions RR-MS patients treated with the two lower doses of HspB5.

<p>RR-MS patients received three bimonthly intravenous administrations of either 7.5 or 12.5 mg HspB5, and mean numbers as well as total volumes of Gd⁺ T1 lesions for the combined groups were evaluated by MRI over the course of 36 weeks. Data show mean values ± standard deviation, as calculated for the full analysis set of patients, including readings for 11 to 14 subjects at different time points. Linear regression analysis was used to evaluate the trends of change over 36 weeks; p values reflect the probability that the deviation of such trends from a horizontal line is a chance event. Arrows indicate times of administration of HspB5.</p

The impact of intravenous HspB5 on active MRI lesions in RR-MS patients.

<p>RR-MS patients received three bimonthly intravenous administrations of either PBS as placebo, or HspB5 at the indicated doses, and mean numbers of Gd⁺ T1 lesions for each group were evaluated by MRI over 36 weeks. Fig 4 shows mean values ± standard deviation, as calculated for the full analysis set of patients, including readings for 7 to 8 subjects at different time points for each group except for the 7.5-mg group, which included readings for 4 to 8 subjects. Linear regression analysis was used to evaluate the trends of change in each group over 36 weeks; p values reflect the probability that the deviation of such trends from a horizontal line is a chance event. Arrows indicate times of administration of placebo or HspB5.</p

alpha B-Crystallin Is a Target for Adaptive Immune Responses and a Trigger of Innate Responses in Preactive Multiple Sclerosis Lesions

We present the first comparative analysis of serum immunoglobulinG reactivity profiles against the full spectrum of human myelin-associated proteins in multiple sclerosis patients and healthy control subjects. In both groups, serum antibodies display a consistent and prominent reaction to αB-crystallin (CRYAB) versus other myelin proteins. As an apparently major target for the adaptive immune system in humans, CRYAB selectively accumulates in oligodendrocytes, but not in astrocytes, or axons in so-called preactive multiple sclerosis lesions. These are clusters of activated HLA-DR-expressing microglia in myelinated normal-appearing white matter with no obvious leukocyte infiltration. They are found in most multiple sclerosis patients at all stages of disease. In these lesion areas, CRYAB in oligodendrocytes may come directly in contact with activated HLA-DR microglia. We demonstrate that CRYAB activates innate responses bymicroglia by stimulating the secretion of leukocyte-recruiting factors, including tumor necrosis factor, interleukin 17, CCL5, and CCL1, and immune-regulatory cytokines such as interleukin 10, transforming growth factor-β, and interleukin 13. Together, these data suggest that CRYAB accumulation in preactive lesions may be part of a reversible reparative local response that involves both oligodendrocytes and microglia. At the same time, however, accumulated CRYAB may represent a major target for adaptive immune responses that could contribute to progression of preactive lesions to a stage of demyelination. © 2010 by the American Association of Neuropathologists, Inc