Progressive remote memory decline coincides with parvalbumin interneuron hyperexcitability and enhanced inhibition of cortical engram cells in a mouse model of Alzheimer’s disease

Reviewer #1 (Public review):

This study presents evidence that remote memory in the APP/PS1 mouse model of Alzheimer's disease (AD) is associated with PV interneuron hyperexcitability and increased inhibition of cortical engram cells. Its strength lies in the fact that it explores a neglected aspect of memory research - remote memory impairments related to AD (for which the primary research focus is usually on recent memory impairments) -which has received minimal attention to date. While the findings are intriguing, the weakness of the paper hovers around purely correlational types of evidence and superficial data analyses, which require substantial revisions as outlined below.

Major concerns:

(1) In light of previous work, including that by the authors themselves, the data in Figure 1 should be complemented by measurements of recent memory recall in order to assess whether remote memories are exclusively impaired or whether remote memory recall merely represents a continuation of recent memory impairments.

(2) Figure 2 shows electrophysiological properties of PV cells in the mPFC that correlate with the behavior shown in Figure 1. However, the mice used in Figure 2 are different than the mice used in Figure 1. Thus, the data are correlative at best, and the authors need to confirm that behavioral impairments in the APP/PS1 mice crossed to PV-Cre (and SST-Cre mice) used in Figure 2 are similar to those of the APP/PS1 mice used in Figure 1. Without that, no conclusions between behavioral impairments and electrophysiological as well as engram reactivation properties can be made, and the central claims of the paper cannot be upheld.

(3) The reactivation data starting in Figure 3 should be analysed in much more depth: a) The authors restrict their analysis to intra-animal comparisons, but additional ones should be performed, such as inter-animal (WT vs APP/PS1) as well as inter-age (12-16w vs 16-20w). In doing so, reactivation data should be normalized to chance levels per animal, to account for differences in labelling efficiency - this is standard in the field (see original Tonegawa papers and for a reference). This could highlight differences in total reactivation that are already apparent, such as for instance in WT vs APP/PS1 at 20w (Figure 3o), and highlight a decrease in reactivation in AD mice at this age, contrary to what is stated in lines 213-214. b) Comparing the proportion of mcherry+ cells in PV- and PV+ is problematic, considering that the PV- population is not "pure" like the PV+, but rather likely to represent a mix of different pyramidal neurons (probably from several layers), other inhibitory neurons like SST and maybe even glial cells. Considering this, the statement on line 218 is misleading in saying that PVs are overrepresented. If anything, the same populations should be compared across ages or groups. c) A similar concern applies to the mcherry- population in Figure 4, which could represent different types of neurons that were never active, compared to the relatively homogeneous engram mcherry+ population. This could be elegantly fixed by restricting the comparison to mCherry+Fos+ vs mCherry+Fos- ensembles, and could indicate engram reactivation-specific differences in perisomatic inhibition by PV cells.

(4) At several instances, there are some doubts about the statistical measures having been employed: a) In Figure 4f, it is unclear why a repeated measurement ANOVA was used as opposed to a regular ANOVA. b) In Supplementary Figure 2b, a Mann-Whitney test was used, supposedly because the data were not normally distributed. However, when looking at the individual data points, the data does seem to be normally distributed. Thus, the authors need to provide the test details as to how they measured the normalcy of distribution.

Minor concerns:

(1) Line 117: The authors cite a recent memory impairment here, as shown by another paper. However, given the notorious difficulty in replicating behavioral findings, in particular in APP/PS1 mice (number of backcrossings, housing conditions, etc., might differ between laboratories), such a statement cannot be made. The authors should either show in their own hands that recent memory is indeed affected at 12 weeks of age, or they should omit this statement.

(2) Pertaining to Figure 3, low-resolution images of the mPFC should be provided to assess the spread of injection and the overall degree of double-positive cells.

Reviewer #2 (Public review):

This study presents a comprehensive investigation of remote memory deficits in the APP/PS1 mouse model of Alzheimer's disease. The authors convincingly show that these deficits emerge progressively and are paralleled by selective hyperexcitability of PV interneurons in the mPFC. Using viral-TRAP labeling and patch-clamp electrophysiology, they demonstrate that inhibitory input onto labeled engram cells is selectively increased in APP/PS1 mice, despite unaltered engram size or reactivation. These findings support the idea that alterations in inhibitory microcircuits may contribute to cognitive decline in AD.

However, several aspects of the study merit further clarification. Most critically, the central paradox, i.e., increased inhibitory input without an apparent change in engram reactivation, remains unresolved. The authors propose possible mechanisms involving altered synchrony or impaired output of engram cells, but these hypotheses require further empirical support. Additionally, the study employs multiple crossed transgenic lines without reporting the progression of amyloid pathology in the mPFC, which is important for interpreting the relationship between circuit dysfunction and disease stage. Finally, the potential contribution of broader network dysfunction, such as spontaneous epileptiform activity reported in APP/PS1 mice, is also not addressed.