Genetic knockdown of Klk8 has sex-specific multi-targeted therapeutic effects on Alzheimer's pathology in mice.

AIMS: Previous work in our lab has identified the protease kallikrein-8 (KLK8) as a potential upstream mover in the pathogenesis of Alzheimer's disease (AD). We showed pathologically elevated levels of KLK8 in the cerebrospinal fluid and blood of patients with mild cognitive impairment or dementia due to AD, and in brains of patients and transgenic CRND8 (TgCRND8) mice in incipient stages of the disease. Furthermore, short-term antibody-mediated KLK8 inhibition in moderate stage disease alleviated AD pathology in female mice. However, it remains to be shown whether long-term reversal of KLK8 overexpression can also counteract AD. Therefore, the effects of genetic Klk8-knockdown were determined in TgCRND8 mice. METHODS: The effects of heterozygous ablation of murine Klk8 (mKlk8) gene on AD pathology of both sexes were examined by crossbreeding TgCRND8 [hAPP+/-] with mKlk8-knockdown [mKlk8+/-] mice resulting in animals with or without AD pathology which revealed pathologically elevated or normal KLK8 levels. RESULTS: mKlk8-knockdown had negligible effects on wildtype animals but led to significant decline of amyloid beta (Aß) and tau pathology as well as an improvement of structural neuroplasticity in a sex-specific manner in transgenics. These changes were mediated by a shift to non-amyloidogenic cleavage of the human amyloid precursor protein (APP), recovery of the neurovascular unit and maintaining microglial metabolic fitness. Mechanistically, Klk8-knockdown improved Aß phagocytosis in primary glia and Aß resistance in primary neurons. Most importantly, transgenic mice revealed less anxiety and a better memory performance. CONCLUSIONS: These results reinforce the potential of KLK8 as a therapeutic target in AD.

Inhibition of excessive kallikrein-8 improves neuroplasticity in Alzheimer's disease mouse model.

We recently identified excessive cerebral kallikrein-8 (KLK8) mRNA and protein levels at incipient stages of Alzheimer's disease (AD) in AD patients and TgCRND8 mice. Additionally, we showed that antibody-mediated KLK8 inhibition exerts therapeutic effects on AD along with enhancing neuroplasticity, resulting in improved spatial memory in mice. Mounting evidence further substantiates an important role of the protease KLK8 in neuroplasticity. In the present study we sought to gain new mechanistic insights in the interplay between KLK8, neuroplasticity and tau phosphorylation in the context of AD. We here demonstrate that KLK8 inhibition increased the number of hippocampal Ki-67 and doublecortin positive, proliferative neuronal progenitor cells in transgenic mice, whereas the same action in wildtypes had no effect. In line with these results, KLK8 inhibition reduced the levels of its pro-proliferative interaction partners KLK6 and protease-activated receptor 2 only in wildtypes, while the levels of its proliferation-supporting substrate neuregulin-1 and the non-complexed form of its complexing-partner phosphatidylethanolamine binding protein 1 were enhanced in both genotypes. Concomitant incubation of beta-amyloid (Aß)-producing primary neurons with KLK8 and its inhibitory antibody increased neurite complexity and soma size. KLK8 inhibition in SH-SY5Y cells or in primary neurons increased levels of the neuroplasticity-supporting KLK8 substrate ephrin receptor B2 (EPHB2) and total tau while decreasing the relative amount of phospho-tau in relation to total tau. KLK8 blockade further enhanced cell proliferation in SH-SY5Y cells. Additional co-incubation with an inhibitory anti-EPHB2 antibody decreased total tau levels and neurite complexity and increased the ratio of phospho-tau/total tau, underlining the key role of EPHB2 on this plastic change. In a reverse in vitro approach, KLK8 induction reduced EPHB2 and total tau and increased the ratio of phospho-tau/total tau, leading to impaired proliferation and neuronal differentiation. These results underline the therapeutic potential of KLK8 inhibition by counteracting plasticity deficits in AD-affected brain.

Higher levels of kallikrein-8 in female brain may increase the risk for Alzheimer's disease.

Women seem to have a higher vulnerability to Alzheimer's disease (AD), but the underlying mechanisms of this sex dichotomy are not well understood. Here, we first determined the influence of sex on various aspects of Alzheimer's pathology in transgenic CRND8 mice. We demonstrate that beta-amyloid (Aß) plaque burden starts to be more severe around P180 (moderate disease stage) in female transgenics when compared to males and that aging aggravates this sex-specific difference. Furthermore, we show that female transgenics suffer from higher levels of neurovascular dysfunction around P180, resulting in impaired Aß peptide clearance across the blood-brain-barrier at P360. Female transgenics show also higher levels of diffuse microgliosis and inflammation, but the density of microglial cells surrounding Aß plaques is less in females. In line with this finding, testosterone compared to estradiol was able to improve microglial viability and Aß clearance in vitro. The spatial memory of transgenics was in general poorer than in wildtypes and at P360 worse in females irrespective of their genotype. This difference was accompanied by a slightly diminished dendritic complexity in females. While all the above-named sex-differences emerged after the onset of Aß pathology, kallikrein-8 (KLK8) protease levels were, as an exception, higher in female than in male brains very early when virtually no plaques were detectable. In a second step, we quantified cerebral KLK8 levels in AD patients and healthy controls, and could ascertain, similar to mice, higher KLK8 levels not only in AD-affected but also in healthy brains of women. Accordingly, we could demonstrate that estradiol but not testosterone induces KLK8 synthesis in neuronal and microglial cells. In conclusion, multiple features of AD are more pronounced in females. Here, we show for the first time that this sex-specific difference may be meditated by estrogen-induced KLK8 overproduction long before AD pathology emerges.

Late running is not too late against Alzheimer's pathology.

In the last decade a vast number of animal studies have produced overwhelming evidence that exercise not only compensates for memory loss by increasing brain plasticity and cognitive reserve but also directly counteracts Alzheimer-like pathology when provided before disease onset or in early disease stages. But so far, there is little knowledge about therapeutic effects of training when started in advanced disease stages. In the present study we show that following seven months of sedentary life style five months of wheel running, started four months after disease onset was still able to mitigate at least some aspects of the full-blown Alzheimer's pathology in TgCRND8 mice. Late running had mild but significant effects on structural plasticity by increasing the dendritic complexity. It further reduced beta-amyloid (Aß) plaque burden and enhanced Aß clearance across the blood-brain barrier, along with attenuating microgliosis, inflammation, oxidative stress, and autophagy deficits, resulting in better memory performance and less agitation. However, unlike early exercise, late running did not affect abnormal amyloid precursor protein metabolism, tau pathology, or angiogenesis. These results allow concluding that it is never too late to counteract Alzheimer's disease with physical training but the earlier the intervention starts, the more pronounced is the therapeutic potential.

Kallikrein-8 inhibition attenuates Alzheimer's disease pathology in mice.

INTRODUCTION: Memory loss and increased anxiety are clinical hallmarks of Alzheimer's disease (AD). Kallikrein-8 is a protease implicated in memory acquisition and anxiety, and its mRNA is known to be up-regulated in AD-affected human hippocampus. Therefore, an involvement of Kallikrein-8 in Alzheimer's pathogenesis is conceivable but remains to be proved. METHODS: We determined the cerebral expression of Kallikrein-8 mRNA and protein during the course of AD in patients and in transgenic mice and tested the impact of Kallikrein-8 inhibition on AD-related pathology in mice and in primary glial cells. RESULTS: Kallikrein-8 mRNA and protein were up-regulated in both species at incipient stages of AD. Kallikrein-8 inhibition impeded amyloidogenic amyloid-precursor-protein processing, facilitated amyloid ß (Aß) clearance across the blood-brain-barrier, boosted autophagy, reduced Aß load and tau pathology, enhanced neuroplasticity, reversed molecular signatures of anxiety, and ultimately improved memory and reduced fear. DISCUSSION: Kallikrein-8 is a promising new therapeutic target against AD.

GluA2 is rapidly edited at the Q/R site during neural differentiation in vitro.

The majority of AMPA receptors in the adult brain contain GluA2 subunits, which can be edited at the Q/R site, changing a glutamine to an arginine within the ion pore. Q/R editing renders AMPARs virtually Ca(2+)-impermeable, which is important for normal AMPA receptor function. Thus, all GluA2 subunits are Q/R-edited in the adult brain. However, it has remained controversial precisely when editing sets in during development. In the present study, we show that GluA2 mRNA is very rapidly Q/R-edited immediately after its appearance, which is after 4.5 days of differentiation from 46C embryonic stem cells (ESCs) to neuroepithelial precursor cells (NEPs). At this time point, most of the GluA2 transcripts were already edited, with only a small fraction remaining unedited, and half a day later all GluA2 transcripts were edited. This can be explained by the observation that the enzyme that Q/R-edits GluA2 transcripts, ADAR2, is already expressed in the cell well before GluA2 transcription starts, and later is not significantly upregulated any more. Editing at another site works differently: The R/G site within the ligand-binding domain was never completely edited at any of the developmental stages tested, and the enzyme that performs this editing, ADAR1, was significantly upregulated during neural differentiation. This confirms previous data suggesting that R/G editing, in contrast to Q/R editing, progresses gradually during development.

Undifferentiated embryonic stem cells express ionotropic glutamate receptor mRNAs.

Ionotropic glutamate receptors (iGluRs) do not only mediate the majority of excitatory neurotransmission in the vertebrate CNS, but also modulate pre- and postnatal neurogenesis. Most of the studies on the developmental role of iGluRs are performed on neural progenitors and neural stem cells (NSCs). We took a step back in our study by examining the role of iGluRs in the earliest possible cell type, embryonic stem cells (ESCs), by looking at the mRNA expression of the major iGluR subfamilies in undifferentiated mouse ESCs. For that, we used two distinct murine ES cell lines, 46C ESCs and J1 ESCs. Regarding 46C ESCs, we found transcripts of kainate receptors (KARs) (GluK2 to GluK5), AMPA receptors (AMPARs) (GluA1, GluA3, and GluA4), and NMDA receptors (NMDARs) (GluN1, and GluN2A to GluN2D). Analysis of 46C-derived cells of later developmental stages, namely neuroepithelial precursor cells (NEPs) and NSCs, revealed that the mRNA expression of KARs is significantly upregulated in NEPs and, subsequently, downregulated in NSCs. However, we could not detect any protein expression of any of the KAR subunits present on the mRNA level either in ESCs, NEPs, or NSCs. Regarding AMPARs and NMDARs, GluN2A is weakly expressed at the protein level only in NSCs. Matching our findings for iGluRs, all three cell types were found to weakly express pre- and postsynaptic markers of glutamatergic synapses only at the mRNA level. Finally, we performed patch-clamp recordings of 46C ESCs and could not detect any current upon iGluR agonist application. Similar to 46C ESCs, J1 ESCs express KARs (GluK2 to GluK5), AMPARs (GluA3), and NMDARs (GluN1, and GluN2A to GluN2D) at the mRNA level, but these transcripts are not translated into receptor proteins either. Thus, we conclude that ESCs do not contain functional iGluRs, although they do express an almost complete set of iGluR subunit mRNAs.