CN111655319A - Treatment of dementia with visual stimulation synchronized with oscillations in the brain - Google Patents

Abstract

Devices, systems, and methods for treating dementia or Alzheimer's disease in a subject in need thereof are disclosed. In one example, a chronic visual stimulus having a frequency of about 30Hz to about 50Hz, more specifically about 40Hz, is non-invasively delivered to the subject to entrain gamma oscillations in multiple brain regions of the subject, including the prefrontal cortex (PFC) and hippocampus. The entrained gamma oscillations modulate neuronal activity across multiple brain regions (e.g., promote functional binding of neural networks at low gamma frequencies) to elicit various neuroprotective effects (e.g., improve amyloid plaques and tau hyperphosphorylation) and reduce neurodegeneration. Chronic visual stimulus-mediated neuronal activity reduces the immune response of microglia and improves aberrantly modified genes and proteins involved in membrane trafficking, intracellular transport, synaptic function, neuroinflammation and DNA damage response. Behavioral changes including enhanced learning and memory are observed.

Description

Treatment of dementia with visual stimulation synchronized with oscillations in the brain

Cross Reference to Related Applications

This application claims priority to each of the following U.S. provisional applications: serial No. 62/570,250 (attorney docket No. MITX-9699/00US) filed on 10/2017 and entitled "neuro rolling effiects OF COMBINED sensing study"; and serial No. 62/570,929 (attorney docket No. MITX-0070/00US) entitled "GAMMAENTRAINMENT BINDS HIGHER ORDER BRAIN REGIONS AND OFFERS NEUROPROTECTION", filed on 11/10/2017. Each of these provisional applications is incorporated herein by reference in its entirety. The present application also claims priority from PCT application No. PCT/US18/51785, entitled "Systems and Methods for predicting, Mitigating, and/or Treating Dementia," filed 2018, 9/19, which is incorporated herein by reference in its entirety.

Statement of government support

The invention was made with U.S. government support under grant number RF1AG054321 awarded by the national institutes of health. The united states government has certain rights in the invention.

Background

Dementia, including Alzheimer's Disease (AD), is a devastating encephalopathy characterized by a decline in brain and cognitive function (Canter et al, 2016; Palop and Mucke, 2016). A variety of factors contribute to the pathogenesis of AD, including amyloid- β deposition, hyperphosphorylated tau accumulation, microglial and astrocyte-mediated inflammation, and neuronal and synaptic loss (Balatore et al, 2007; Huang and Mucke, 2012; Jacobsen et al, 2006; Meyer-Luehmann et al, 2008; Oakley et al, 2006; Ulland et al, 2017; Yoshiyama et al, 2007).

Recent studies have increasingly studied physiological aspects of AD lesions such as neuronal hyperexcitability, interneuron dysfunction, inhibition/excitation balance shifts, epileptic discharges, and changes in network oscillations (Canter et al, 2016; Holth et al, 2017; ia et al, 1999; Palop and Mucke, 2010; Palop and Mucke, 2016; Verret et al, 2012). These findings are consistent with the network abnormalities observed in human AD (Guillon et al, 2017; Koenig et al, 2005; Ribary et al, 1991; Stam et al, 2002). Recent studies in mouse models of AD have emphasized that these changes occur in the pre-symptomatic phase (Gillespie et al, 2016; Iaccarino et al, 2016). Changes in neural activity have previously been shown to affect AD pathology such as amyloid- β and tau accumulation in several mouse models (Bero et al, 2011; Wu et al, 2016; Yamada et al, 2014). In view of these observations, various approaches have been taken to investigate whether manipulating neuronal oscillations could effectively improve AD lesions (Iaccarino et al, 2016; Martinez-Losa et al, 2018; Verret et al, 2012).

In particular, it has been found that oscillations in the gamma band (-30-90 Hz) are reduced in a variety of mouse models of AD, including hAPP-J20, ApoE4, 5XFAD, but are also particularly evident in human AD patients (Gillespie et al, 2016; Guillon et al, 2017; Iaccarino et al, 2016; Koenig et al, 2005; Ribary et al, 1991; Stam et al, 2002; Verret et al, 2012). In view of this, some recent studies have been directed to gamma oscillation, and their findings suggest that this may represent a promising strategy for alleviating AD lesions.

In one previous approach, gamma deficiency was alleviated and both epileptiform activity and cognitive decline in hAPP-J20 mice were reduced by expressing the voltage-gated sodium channel subunit nav1.1 in parvalbumin positive (PV +) interneurons, or by increasing gamma oscillations by brain transplantation of nav 1.1-overexpressed interneuron progenitor cells (Martinez-Losa et al, 2018; Verret et al, 2012).

In the second approach, optogenetic activation of PV + interneurons at 40Hz, which have been shown to cause strong gamma frequency oscillations (Cardin et al, 2009; Sohal et al, 2009), was found to reduce amyloid burden and enhance the morphological transformation of microglia in 5XFAD mice (Iaccarino et al, 2016). This non-invasive approach using 40Hz visual stimulation was equally effective in reducing amyloid burden and altering microglia in the visual cortex of 5XFAD mice (Iaccarino et al, 2016). However, in this previous study, amyloid levels in the visual cortex returned to baseline 24 hours after one hour of acute visual stimulation. However, this earlier study found that extending visual stimulation from one hour to one hour per day for seven days not only reduced amyloid levels (soluble and insoluble forms of a β 1-40 and a β 1-42), but also reduced plaque lesions in the visual cortex of 6 month old 5XFAD mice (Iaccarino et al, 2016). In addition, visual stimuli affect a variety of cell types, including neurons and microglia, to reduce the production of a β and enhance its clearance in 5XAFD mice, respectively.

Disclosure of Invention

As disclosed in U.S. patent application serial No. 15/360,637 filed on 23/11/2016 (hereby incorporated by reference herein in its entirety) and entitled "SYSTEMS AND METHODS FOR PREVENTING, MITIGATING, AND/OR TREATING DEMENTIA," inducing synchronized gamma oscillations in the brain via visual (as well as auditory and/OR tactile) stimulation results in amyloid burden reduction and morphological changes in some brain regions. However, the inventors have recognized and appreciated that there remains a need for systems and methods for treating dementia and alzheimer's disease that address all-circuit disorders affecting multiple brain centers that are important responsible for learning and memory as well as other higher brain functions.

In the present disclosure, the inventive methods and apparatus for entraining gamma oscillations in a subject's brain by chronic non-invasive visual stimulation, referred to herein as "gamma entrainment with sensory visual stimulation" (GENUS), beyond the visual cortex to multiple other brain regions (e.g., hippocampus, somatosensory cortex, and prefrontal cortex), while also enhancing low gamma coherence across these multiple brain regions, have been demonstrated. In addition, chronic GENUS reduced the neurodegeneration in 5XFAD, P301S and CK-P25 mice and had a clear neuroprotective effect across multiple brain regions. The data collected underscores how GENUS-mediated modulation of neuronal activity across brain regions affects genes and proteins involved in intracellular transport and synaptic function in degenerated neurons. These results establish a link between GENUS-driven gamma oscillations, functional integration of neural networks across multiple brain regions, neuroprotection, and behavioral performance of the subject.

In summary, one embodiment of the invention relates to a method for treating dementia or alzheimer's disease in a subject in need thereof, the method comprising: A) non-invasively delivering to a subject a chronic visual stimulus having a frequency of about 30Hz to about 50Hz to entrain synchronized gamma oscillations in multiple brain regions of the subject, including at least the prefrontal cortex (PFC) and hippocampus of the subject.

Another invention embodiment relates to a method for treating dementia or alzheimer's disease in a subject in need thereof, the method comprising: A) non-invasively delivering a chronic visual stimulus having a frequency of about 30Hz to about 50Hz to a subject to entrain synchronized gamma oscillations in multiple brain regions of the subject and improve aberrantly modified genes and proteins in degenerating neurons in the multiple brain regions of the subject.

Another invention embodiment relates to a method for treating dementia or alzheimer's disease in a subject in need thereof, the method comprising: A) non-invasively delivering to a subject a chronic visual stimulus having a frequency of about 30Hz to about 50Hz to simultaneously entrain synchronized gamma oscillations in multiple brain regions of the subject to significantly increase gamma coherence between the multiple brain regions of the subject having a frequency between 30Hz to 50 Hz.

It should be understood that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided that such concepts do not contradict each other) are considered a part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are considered part of the inventive subject matter disclosed herein. It should also be understood that terms explicitly employed herein that may also appear in any disclosure incorporated by reference should be given the most consistent meaning to the particular concepts disclosed herein.

Other systems, processes, and features will be or become apparent to one with skill in the art upon examination of the following figures and detailed description. It is intended that all such additional systems, processes, and features be included within this description, be within the scope of the present invention, and be protected by the accompanying claims.

Drawings

This patent or application document contains at least one drawing executed in color.

Those skilled in the art will appreciate that the drawings are primarily for illustrative purposes and are not intended to limit the scope of the inventive subject matter described herein. The figures are not necessarily to scale; in some instances, aspects of the subject matter disclosed herein may be shown exaggerated or enlarged in the drawings to facilitate an understanding of various features. In the drawings, like reference characters generally refer to the same features (e.g., functionally similar and/or structurally similar elements), for example.

Fig. 1A-1J illustrate visual stimuli according to the disclosed inventive concepts entraining gamma oscillations beyond the visual cortex in multiple brain regions of a subject.

Fig. 2A-2F show that chronic 40Hz (but not 80Hz) visual flicker stimulation according to the disclosed inventive concepts reduces amyloid plaques beyond the visual cortex in a subject.

Fig. 3A-3J show that chronic visual stimulation according to the disclosed inventive concepts improves the pathology associated with alzheimer's disease and significantly reduces or prevents neurodegeneration in a subject.

Fig. 4A-4Q show that chronic visual stimuli according to the disclosed inventive concepts reduce the inflammatory response of microglia in a subject.

Fig. 5A-5I illustrate that chronic visual stimuli according to the disclosed inventive concepts alter synaptic function and intracellular transport in neurons.

Figures 6A-6I illustrate that chronic visual stimuli according to the disclosed inventive concepts alter behavior in multiple subject models of alzheimer's disease.

Fig. 7A-7I show that chronic visual stimulation according to the disclosed inventive concepts entrains gamma oscillations beyond the visual cortex in a mouse model of neurodegeneration.

Fig. 8A-8I show that chronic visual stimuli according to the disclosed inventive concepts reduce AD-associated lesions beyond the visual cortex in 5XFAD mice.

Fig. 9A-9G show that chronic visual stimulation according to the disclosed inventive concept improves AD-associated lesions in P301S and CK-P25 mice.

Fig. 10A-10N show that chronic visual stimulation according to the disclosed inventive concepts alters microglia, improving intracellular transport and synaptic transmission in neurons.

Fig. 11A-11J illustrate behavioral characterization of the impact of acute and chronic visual stimuli on a subject according to the disclosed inventive concepts.

Fig. 12A-12C show that chronic visual stimulation at 80Hz does not affect the Morris water maze of 5XFAD mice according to the disclosed inventive concepts.

Detailed Description

The following is a more detailed description of various concepts and embodiments thereof related to systems and methods for preventing, alleviating and/or treating dementia through visual stimuli that combine higher brain regions, reduce neurodegeneration and neuroinflammation, and improve cognitive function. It should be appreciated that the various concepts introduced above and discussed in greater detail below may be implemented in a variety of ways. Examples of specific embodiments and applications are provided primarily for illustrative purposes to enable those skilled in the art to practice the embodiments and alternatives apparent to those skilled in the art.

The figures and example embodiments described below are not intended to limit the scope of the embodiments to a single embodiment. Other embodiments are possible by interchanging some or all of the elements described or shown. Further, where certain elements of the disclosed example embodiments may be partially or fully implemented using known components, in some cases only those portions of such known components that are necessary for an understanding of the present embodiments are described, and detailed descriptions of other portions of such known components are omitted so as not to obscure the present embodiments.

In this disclosure, we demonstrate that the inventive technique involving treating a subject with chronic non-invasive visual stimuli, referred to herein as "gamma entrainment with sensory visual stimuli" (GENUS), addresses symptoms associated with dementia, including Alzheimer's Disease (AD), and affects AD lesions in brain regions outside the visual cortex. In an illustrative example, we demonstrate the role of chronic vision GENUS in various neurodegenerative mouse models, including Tau P301S, CK-P25, and older 5XFAD mice. We observed that chronic vision GENUS entrains gamma oscillations in multiple brain regions (including higher brain regions) and induces functional binding at low gamma frequencies across these brain regions. In several mouse models of neurodegenerative diseases we tested, we found that chronic visual GENUS ameliorates a variety of AD-associated pathologies, including amyloid plaques, tau hyperphosphorylation, and brain atrophy, preventing neuronal and synaptic density loss in multiple higher brain regions. Transcriptomic and proteomic analysis profiles demonstrated that abnormally modified genes and proteins involved in membrane trafficking, intracellular trafficking, and synaptic function in degenerated neurons of P301S and CK-P25 mice were improved or repaired with chronic GENUS and microglial immune responses were reduced. In view of these extensive neuroprotective effects, we further investigated the impact of chronic daily GENUS on cognitive function and demonstrated improved performance of behavioral tasks in various mouse models of AD. In summary, our results underscore the neuroprotective efficacy of visual GENUS in treating dementia (including AD) subjects.

As will be described in detail below, several studies were conducted to assess the effects on multiple brain regions of chronic visual stimuli. The visual stimulus typically has a frequency of about 30Hz to 50Hz, with particular attention paid to the fact that the frequency of the visual stimulus is at or about 40 Hz. In some examples, a Light Emitting Diode (LED) based device is employed to deliver the visual stimulus and the LED based device is driven in a square wave current pattern with a 50% duty cycle. However, it should be understood that various types of devices (other than LED-based devices) may be employed to effectively deliver visual stimuli at various frequencies within the ranges described herein. Additionally, waveforms other than square wave form and duty cycles other than 50% may be employed to effectively produce visual stimuli at various frequencies within the ranges described herein.

In addition, various treatment regimens were employed in some of the studies described herein, including a subject exposure time of 1 hour per day for visual stimuli and a subject exposure period of 7 days, 22 days (about 3 weeks) and 42 days (about 6 weeks). However, it is understood that other subject exposure times and subject exposure periods may be employed to deliver visual stimuli at various frequencies within the ranges described herein and to effectively treat dementia, including alzheimer's disease. For example, exposure times of greater than 1 hour per day (e.g., in multiple 1 hour increments, delivered in shorter increments, or in longer increments), and/or exposure periods of less than three weeks, between three and six weeks, and greater than six weeks, can be used in various combinations and permutations to effectively treat dementias, including alzheimer's disease.

Below we first provide an experimental abstract and respective observations, followed by further details of the experimental protocol to illustrate the efficacy of visual GENUS on dementia (including alzheimer's disease) subjects.

Visual GENUS affects the higher brain areas

Our primary goal was to determine whether a 40Hz visual stimulus could modulate neuronal activity in brain regions outside the visual cortex, or whether it was confined to the visual cortex, by performing C-Fos immunohistochemical staining as a marker of neuronal activation in wild-type C57Bl/6J mice (fig. 1A). Visual stimuli were delivered at a specific frequency with a custom LED device at a 50% duty cycle (e.g., 12.5ms on and 12.5ms off for 40Hz stimulation) (Iaccarino et al, 2016; Singer et al, in print). Mice were acclimated for 3 days and then exposed to a visual stimulus of 40Hz for 1 hour. They were then returned to their cages for 1 hour, then sacrificed, brain tissue collected, sectioned, and labeled with c-Fos antibody. We found that 40Hz visual stimulation resulted in a significant increase in c-Fos positive neurons in the visual cortex (V1) (fig. 1A-1B), and also noted a significant increase in the somatosensory cortex (SS1), hippocampal CA1, and Cingulate Cortex (CC) region of the prefrontal cortex (fig. 1A-1B).

To understand how a 40Hz visual stimulus can alter ongoing neural activity in these brain regions, we implanted a custom-made microactuator (tungsten wire electrode; see methods for details) into C57Bl/6J mice to simultaneously record Local Field Potentials (LFPs) of the cingulated gyrus regions of V1, SS1, CA1 and the prefrontal cortex (PFC) of free-moving mice. Mice were confined in a small GENUS box (8x 8 inches) to reduce exploratory behavior and were simultaneously recorded with Local Field Potentials (LFPs) from V1, SS1, CA1, and prefrontal cortex (PFC) when they were exposed to a 40Hz visual stimulus of initial occlusion and then 10 minutes each time the occlusion was released (fig. 1A, fig. 7A). Acute 40Hz visual stimulation significantly increased the power of low gamma oscillations around 35-45Hz, with a peak frequency at 40Hz in V1 (fig. 1C-1D), with a small but significant increase in CA1 (fig. 1C-1D), consistent with previous findings in head-constrained mice (Iaccarino et al, 2016). A small but significant increase in gamma power was also observed in SS1 and PFCs (fig. 1C to 1D), consistent with the increase we observed in C-Fos positive cells in these regions (fig. 1B).

To assess whether these visual stimuli-induced oscillations were able to entrain neuronal firing in brain regions downstream of the visual cortex, we implanted quadrupoles against hippocampal CA1 into a group of C57Bl/6J mice to record single unit activity. Under baseline conditions where light was occluded, the CA1 pyramidal cells showed strong phase lock for low γ (-35-45 Hz) and preferentially discharged at the peak, consistent with previous reports (Bragin et al, 1995; Middleton and McHugh, 2016) (FIG. 1E). At a visual stimulus of 40Hz, although the preferred phase was not altered, a more firm phase lock was induced for each neuron, quantified as an increased average resulting length across the population (MRL; between blocking stimulus and 40Hz visual stimulus, P ═ 0.01) (fig. 1F), suggesting that gamma frequency visual stimuli were used to drive the hippocampal neuron population in a more organized manner in time.

To test the applicability of GENUS to the impaired neuronal system, we used several mouse models of neurodegeneration to verify whether we could cause gamma oscillations by visual stimulation, as observed in C57Bl/6J animals. We used a CK-p25 mouse strain in which expression of Cdk5 activator p25 was driven in an inducible manner by an excitatory neuron-specific CaMKII α promoter (CaMKII α promoter-tTA x TetO-p25+ GFP) (Cruz et al, 2003). CK-p25 exhibited progressive neuronal and synaptic loss following withdrawal of doxycycline from the diet, with cognitive impairment, which became severe by p25 induction up to 6 weeks (Cruz et al, 2003). Consistent with these findings, LFP recordings in vivo from CK-p25 mice showed a large reduction in gamma spectral power at 35-45Hz in V1, CA1 and PFC compared to control mice 6 weeks after induction (fig. 7B to 7C). Despite these changes, visual stimulus at 40Hz was still able to significantly enhance gamma oscillations in V1, CA1, and PFC (fig. 7C). We further examined Tau P301S mice, which express high levels of protein Tau associated with microtubules by humanized mutants and have Tau aggregates associated with frontotemporal dementia at 5 months of age (Yoshiyama et al, 2007). 8-month old P301S mice with synaptic and neuronal loss and cognitive deficits exhibited enhanced gamma power when visually stimulated at 40Hz in V1 and PFC (fig. 7D-7E). These results indicate that GENUS is sufficient to entrain gamma oscillations in AD mice, regardless of the specific pattern of neuronal damage in these mice.

To ensure that gamma oscillations can be entrained even after multiple exposures to visual stimuli over extended periods of time, we used a chronic GENUS protocol, exposing C57Bl/6J and p25 induced CK-p25 mice to 40Hz visual stimuli for 42 days (1 hour/day) (fig. 7F, fig. 7H). LFP recorded from V1, SS1, CA1 and PFC on day 43 showed significantly enhanced 40Hz γ power across all regions of C57Bl/6J mice, consistent with a single experimental acute GENUS application (fig. 7F). In a similar manner, CK-p25 mice also resulted in low γ power increases in V1, CA1 and PFC on day 43 (now 85 days post induction of p25) (fig. 7H). These results, along with the c-Fos immunostaining data, indicate that acute and chronic 40Hz visual stimuli recruit not only the visual cortex, but also other higher-order cortex, including CA1, SS1, and PFC.

Since 40Hz visual stimulation (acute and chronic) enhances the 40Hz power of V1, SS1, CA1, and PFC simultaneously, we also demonstrate that GENUS can coordinate neuronal activity between the visual cortex and these other higher brain structures. We calculated the coherence across these brain regions with and without light being occluded during 40Hz visual stimulation using a Weighted Phase Lag Index (WPLI) method, which minimizes potential contamination by volume conduction (Vinck et al, 2011). We analyzed LFP in C57Bl/6J mice across electrode pairs located in V1-CA1, V1-SS1, V1-PFC, CA1-SS1, and CA 1-PFC. With and without light being blocked, there is no significant difference in average speed during the 40Hz visual stimulus, eliminating any potential difference in motor activity. Acute 40Hz visual stimulation significantly increased the 30-50Hz low gamma coherence (compared to the occluded light period) between the visual cortex and other examined brain regions, particularly between V1-CA1, V1-SS1 and V1-PFC (fig. 1G, fig. 1H).

To evaluate the long-term effects of chronic GENUS on coordinated neuronal oscillation across broader brain structures, we applied WPLI analysis to LFP collected from V1, SS1, CA1 and PFC of C57Bl/6J and p 25-induced CK-p25 mice exposed to visual stimuli for 1 hour/day for 42 days. We observed a significant increase in 30-50Hz low γ WPLI between V1 and CA1 (fig. 7G) and between V1-SS1, V1-PFC and CA1-PFC during 40Hz visual stimulation (fig. 7G) compared to photoblocking conditions. Similarly, CK-p25 mice also showed a significant increase in 30-50Hz low γ WPLI between V1-CA1, CA1-PFC and V1-PFC after chronic GENUS compared to photoblocking conditions (fig. 7I). Overall, these data indicate that 40Hz visual stimulation enhances local (40Hz entrainment) and intercoordinated regional neurooscillatory activity in multiple superior cortex of mice (30-50Hz low γ WPLI). However, it is important to determine whether the changes we observed are specific to low gamma frequency stimulation. To test this, we exposed C57Bl/6J mice to 80Hz visual stimulation delivered at a 50% duty cycle to ensure that the mice received similar light intensity and exposure duration as our 40Hz stimulation experiment (fig. 1I). Compared to the pre-stimulus period, we observed no significant change in the 80Hz spectral power in the visual cortex during the 80Hz visual stimulus (here we observed the maximum power increase for 40Hz GENUS) (fig. 1I, fig. 1J).

Chronic GENUS ameliorates amyloid plaques outside the visual cortex

Iaccarino et al (2016) demonstrated that acute visual GENUS reduced amyloid levels in V1 in pre-symptomatic young 5XFAD mice, while 7-day visual GENUS not only reduced amyloid levels, but also improved amyloid plaques in 6-month old 5XFAD mice with more advanced lesions. Therefore, we aimed to investigate whether 11-month old 5XFAD mice affected V1 under 7-day visual stimulation at 40Hz, as well as amyloid plaque lesions in CA1, SS1 and PFC. To achieve this, we introduced mice into GENUS stimulation cages (fig. 8A), visually stimulated them at 40Hz or 80Hz (which deliver similar amounts of light to 40Hz, but without entrainment of gamma oscillations in V1 (fig. 1I, fig. 1J)), 1 hour/day for 7 days, and examined for amyloid plaque burden (fig. 2A). We seen a reduction in amyloid plaques in the visual cortex after 7 days under 40Hz visual stimulation (fig. 2A, fig. 2B), but no difference was observed in both SS1 and CA1 compared to non-stimulated mice (fig. 2A, fig. 2B). Consistent with the LFP analysis, 5XFAD mice under 80Hz visual stimulation showed no change in amyloid plaque burden in both V1 and CA1, but a modest increase in SS1 (fig. 2A, 2B). These results show that the reduction of amyloid plaques after 7 days of visual GENUS is limited to V1 and is specific to 40Hz visual stimuli.

Next, we extended the GENUS regimen to 22 days, starting with 9 month old 5XFAD mice, and quantified amyloid plaques at approximately 10 months of mouse age (fig. 2C). We found that 22-day GENUS significantly reduced the intensity and number of amyloid plaques in V1 (fig. 2C, 2D and 8B-8D), consistent with a reduction in amyloid plaques after 7-day GENUS in 6-month-old 5XFAD (Iaccarino et al, 2016) and 11-month-old 5XFAD mice (fig. 2A, 2B). Importantly, 22 days of GENUS were sufficient to reduce plaque intensity and number in the SS1, CA1, and CC regions of the prefrontal cortex (fig. 2D, 8B-8D). This effect was again specific to 40Hz stimulation, as the 22 day 80Hz stimulation did not alter the amyloid plaques in V1, SS1 and CA1 of 5XFAD mice compared to non-stimulated 5XFAD mice (fig. 2E, fig. 2F).

We chose to apply long-term GENUS to 5XFAD mice starting from 9 months of age, since previous studies have shown that progressive loss of neuronal and synaptic markers begins at approximately 9 months of age in the 5XFAD model (Oakley et al, 2006). Consistent with these studies, we observed a significant decrease in neuronal counts in CA1 and CC in 10-month old 5XFAD mice compared to age-matched wild-type litters (fig. 8C, 8E). In contrast, 5XFAD mice receiving 22 days of GENUS showed significantly reduced neuronal loss compared to non-stimulated mice (fig. 8E) in addition to reducing amyloid burden (fig. 2C, 2D and fig. 8B-8D). Similarly, while we observed significant loss of the basoon (synaptic marker protein) site in CA1 and CC of non-stimulated 5XAFD mice, chronic GENUS at 22 days reduced synaptic loss (fig. 8F, 8G). Reduced neurodegeneration and amyloidosis after chronic GENUS were not the result of altered APP transgene expression, as we did not detect differences in expression of full-length APP protein in mice receiving GENUS compared to non-stimulated controls (fig. 8H, fig. 8I). These results demonstrate that chronic vision GENUS results in a reduction in amyloid plaque burden in multiple brain regions of 5XFAD mice, as well as a reduction in neuronal and synaptic loss.

Chronic GENUS reduces neurodegeneration

To further explore the potential of chronic GENUS in affecting disease lesions and reducing neuronal loss, as seen in 5XFAD mice (fig. 8C, fig. 8E), we next investigated a mouse model of Tau P301S and CK-P25 neurodegeneration. At 8 months of age, Tau P301S mice showed marked neuropathy (Yoshiyama et al, 2007). Therefore, we studied a group of Tau P301S mice and performed them for 22 days of 1 hour/day without stimulation or GENUS starting at 7 months of age and examined their Tau phosphorylation levels and Tau-associated lesions at approximately 8 months of age (fig. 3A to 3E and fig. 9A, 9B). We observed that Tau phosphorylation at residues S202/T205 was higher in V1, SS1, CA1 and CC in Tau P301S mice compared to wild type inbred litters (wild type inbred) (fig. 3A, fig. 3B). However, Tau P301S mice receiving chronic GENUS had a significantly reduced degree of S202/T205Tau phosphorylation compared to non-stimulated P301S mice (fig. 3A, 3B). In AD and P301S mice, tau proteins were hyperphosphorylated at multiple residues (Hanger et al, 2007; Wang et al, 2013; Foidl and Humpel, 2018; Kimura et al, 2018), and therefore the degree to which GENUS stimulation can affect tau phosphorylation was examined using the unbiased Ser/Thr (S/T) phosphorylation proteomics method. Compared to wild type inbred litters, we identified over-phosphorylated 46S/T residues and a dephosphorylated single residue (S451) in Tau P301S mice (fig. 3C). Our analysis also showed that chronic GENUS decreased phosphorylation of tau at 6S/T sites and increased phosphorylation of S451, suggesting that GENUS affects tau phosphorylation at multiple sites (fig. 3C).

Next, we characterized the neuronal loss of Tau P301S mice, and as previously reported, they showed a significant reduction in the number of neurons in V1, CA1, SS1 and CC, as quantified by the number of NeuN-positive cells (fig. 3D, fig. 3E). Tau P301S mice, which received GENUS for 22 days from 7 months of age (time point at which neuronal loss began), showed significantly reduced neuronal loss in all brain regions we examined compared to the non-stimulated control (fig. 3D, fig. 3E). Next, we examined brain weight and lateral ventricle size, and observed no difference between wild-type non-stimulated and GENUS-stimulated P301S mice (fig. 9B).

Next, we turned to the CK-p25 model as they showed AD-like pathological features such as brain atrophy, cortical contraction and abnormal expansion of the ventricles 6 weeks after p25 induction (Cruz et al, 2003). To examine the extent to which chronic GENUS can ameliorate these abnormalities, we simultaneously induced p25 in CK-p25 mice for 6 weeks, while also exposing them to 1 hour of GENUS per day (fig. 3F). Non-stimulated CK-p25 mice showed significantly reduced brain weight and cortical thickness compared to CK inbred littermates (CaMKII alpha promoter-tTA; Cruz et al, 2003) (FIG. 3G). Chronic GENUS during p25 induction significantly reduced cortical thinning but did not significantly alter brain weight compared to non-stimulated CK-p25 mice (fig. 3G and fig. 9D). Cortical contraction was closely associated with ventricular dilatation in human AD and CK-p25 transgenic models (Cruz et al, 2003), and we also observed a significant reduction in ventricular dilatation in CK-p25 mice receiving chronic GENUS (fig. 3H and fig. 9E). Furthermore, CK-p25 mice with chronic GENUS during induction of p25 also had significantly less neuronal loss in the CC region of V1, SS1, CA1 and PFC compared to the non-stimulated control (fig. 3I, fig. 3J). It has previously been reported that DNA damage in the form of Double Strand Breaks (DSBs) represents an early marker of neurodegeneration in CK-p25 mice (Kim et al, 2008). Consistent with this finding and our reduction in GENUS-mediated neuronal death, we also observed a significant reduction in DSBs in V1, SS1, and CA1, as analyzed by the number of cells positive for the putative DSB marker γ H2AX (fig. 9G). These neuroprotective effects of chronic GENUS were not induced by altering transgene expression in the mutants, as there was no difference in total tau and P25 expression between GENUS and the non-stimulated group (in P301S and CK-P25 mice, respectively) (fig. 9A, 9C, 9F). Taken together, these findings confirm that chronic GENUS has neuroprotective effects in P301S and CK-P25 mouse models with severe neurodegeneration.

Chronic GENUS reduces inflammatory responses

We also demonstrated that the reduced neurodegeneration we observed in Tau P301S and CK-P25 mice can be mediated in part by a beneficial microglia response following chronic GENUS. We performed unbiased RNA sequencing on the visual cortex of P301S tau and CK-P25 mice that received GENUS stimulation for 22 days and 42 days, respectively, as well as non-stimulated P301S and CK-P25 mice and corresponding wild-type controls (fig. 4A). Visual cortex was dissected, then enzymatically digested, microglia recognized by CD11B and CD45 immunostaining, then isolated using Fluorescence Activated Cell Sorting (FACS) as described previously (Mathys et al, 2017) (fig. 10A, fig. 10B). We isolated 35,000 microglia from each mouse, separately extracted total RNA from each mouse, and examined the quality of the RNA prior to RNA sequencing. An average of 28.69 million reads were obtained per sample, with 89.42% aligned.

RNA sequencing showed that microglia from non-stimulated CK-p25 mice had 2333 up-regulated genes compared to the inbred CK mice (fig. 4A). Next, we performed Gene Ontology (GO) analysis and observed that the up-regulated genes involved protein synthesis, ribosome regulation and immune responses (including viral immune response, antigen presentation and immune response regulation), consistent with previous reports (Mathys et al, 2017) (fig. 4B). By comparison, 2019 down-regulated genes were identified that were primarily associated with cell migration, cell morphogenesis, and vasculature development (fig. 4B). After chronic GENUS, 355 genes in CK-p25 mice were up-regulated (compared to non-stimulated CK-p25) and associated with protein synthesis, mitotic cell cycle regulation, membrane trafficking, and vesicle-mediated transport, while 515 down-regulated genes were associated primarily with gtpase activity, proteolysis, and immune responses, including MHC-1 mediated antigen processing presentation and immune adaptability (fig. 4C). These results indicate that chronic GENUS significantly affected microglial function in CK-p25 mice, rendering them less inflammatory and potentially more capable of phagocytosis, migration, and protein degradation.

In non-stimulated Tau P301S mice, we found a total of 331 up-regulated genes and 292 down-regulated genes relative to their wild type inbred litters (fig. 10C). Gene Ontology (GO) analysis correlated up-regulated genes with protein synthesis and inflammatory/immune responses, while down-regulated genes were more associated with cytoskeletal organization, cell migration, and brain development (fig. 10D). Microglia obtained from Tau P301S mice after chronic GENUS (22 days) showed 238 up-regulated and 244 down-regulated genes compared to non-stimulated Tau P301S mice. Here, up-regulated genes are associated with cellular catabolic proteolysis, cell migration, cell morphogenesis and membrane trafficking, while down-regulated genes are involved in gene expression, translation initiation and interferon response (fig. 10C and 10D). Thus, CK-P25 mice with chronic GENUS showed significantly similar transcriptomic changes as Tau P301S mice with chronic GENUS. Taken together, our microglia-specific transcriptomics analysis indicates that chronic GENUS can morphologically transform microglia, enhance protein degradation and reduce microglia-mediated immune responses, without relying on specific transgenic models (P301S or CK-P25) to generate disease states.

To further validate these findings, we used brain sections from CK-P25 and Tau P301S mice after chronic GENUS (CK-P25: 1 hour/day for 42 days during P25 induction; Tau P301S: 22 days) and from their respective non-stimulated and inbred control groups for immunohistochemical staining. A CK-p25 microglia response has been previously described, in which early responses are characterized by increased proliferation, while late responses are characterized by an elevation of the MHC class II and interferon pathways (Mathys et al, 2017). We first performed immunohistochemistry and 3-dimensional rendering using the microglia-specific marker Iba1, which showed a significant increase in the number of microglia and a wide variation in morphology in V1 of 6-week-induced CK-p25 mice (fig. 4D to fig. 4I and fig. 10E). Microglia in CK-p25 animals showed no significant difference in cell body volume as a whole compared to the control group (fig. 4F), but many microglia showed a more complex "plexiform" branching pattern (arrows) (fig. 4D; lower middle panel), which is associated with axonal and terminal synaptic degeneration (Jensen et al, 1994; Jorgensen et al, 1993). We also noted that most microglia showed elongated rod-like bodies without polarization process (arrows) (fig. 4D, fig. 4I), a phenotype known to exist after diffuse brain injury in rats and human subjects (Bachstetter et al, 2017; Taylor et al, 2014). In addition, despite the reduced treatment volume compared to the pure CK control (fig. 4G), the microglia in CK-p25 were physically closer to each other as analyzed by measuring the minimum distance between the microglia (fig. 4D, fig. 4H). This indicates a loss of its area, and is in contrast to the resting state, where each microglia cell typically has its own occupied area with little overlap between adjacent areas (Del Rio-Hortega, 1932; Nimmerjahn et al, 2005).

Chronic GENUS resulted in a significant reduction in the amount of microglia in CK-p25 mice compared to non-stimulated mice, although it remained higher than pure CK mice (fig. 4D, fig. 4E). The total volume of microglia after chronic GENUS showed less contraction, so that there was no significant difference compared to CK-p25 and the CK pure group (fig. 4G). Importantly, the minimal distance between microglia after chronic GENUS was comparable to that in pure CK animals (fig. 4D, fig. 4H), indicating that chronic GENUS retained areas of microglia. Finally, in CK-p25 animals with chronic GENUS, although the total volume of rod-shaped microglia remained significantly higher than the pure CK group, its increase was significantly reduced (fig. 4I). Next, we characterized Tau P301S microglia and found that there was a trend of increased microglia in P301S, but not statistical significance in our Iba1 immunostaining (fig. 4K, fig. 4L). There was also no difference in total volume of microglastids in P301S compared to wild type inbred mice (fig. 4K, fig. 4M). However, the total volume of microglial processes in Tau P301S mice after chronic GENUS was significantly different from that of non-stimulated P301S mice and comparable to wild-type inbred litters (fig. 4K, fig. 4N). These results underscore the range of microglial states at baseline and under disease conditions and generally indicate the role of chronic GENUS in alleviating disease-associated microglial morphological dysfunction in P301S and CK-P25 mice.

Next, we examined GO terminology, showing that immune response is affected by chronic GENUS. Immunohistochemistry using antibodies specific for interferon response gene CD40 showed significant elevation in CK-p25 mice compared to naive CK mice (fig. 4D, fig. 4J), consistent with previous reports (Mathys et al, 2017). Chronic GENUS resulted in a significant decrease in CD40 signal intensity in CK-p25 mice, although it remained significantly higher than the purebred CK control (fig. 4D, fig. 4J). Next, we examined another immune response gene, C1q (classical complement component), which was significantly up-regulated in our microglia-specific RNA sequencing experiments and was previously associated with synaptic loss in AD mouse models (Hong et al, 2016). Immunohistochemistry for C1Q showed increased signals in V1 in unstimulated CK-P25 and Tau P301S mice compared to their respective inbred control litters (fig. 4O, fig. 4P, fig. 4Q). Chronic GENUS significantly reduced the increase in C1q in CK-P25 mice, although the C1q intensity remained above the true breed CK level (fig. 4O, fig. 4P). In P301S mice, chronic GENUS attenuated the increase in C1Q, making no significant difference between wild-type inbred mice and non-stimulated P301S mice (fig. 4O, fig. 4Q). In summary, our immunohistochemical results consistently indicate that chronic GENUS contributes to a reduction in microglial immune response.

Chronic GENUS alters the regulation of synaptic transmission and intracellular transport in neurons

Next, we isolated NeuN positive (NeuN +) neuronal nuclei and performed unbiased RNA sequencing analysis to investigate the effect of chronic GENUS on gene expression in CK-P25 mice and V1 neurons of Tau P301S (fig. 5A, 5B, and 10F-10G). After chronic GENUS (CK-P25: P25 induction for 42 days; Tau P301S: 22 days), the visual cortex was harvested from the mice. Subsequently, 100,000 neuronal nuclei were sorted by FACS into lysis buffer, followed by extraction and sequencing of RNA (fig. 5A, 5B). Consistent with our findings on brain weight and the quantification of NeuN and cortical thinning by using immunohistochemistry (fig. 3G, fig. 3I, and fig. 9D), we found that the percentage of NeuN + nuclei was significantly reduced in non-stimulated CK-p25 mice (81.56 ± 3.29) when compared to the inbred CK litter (100 ± 3.87), which was not apparent in CK-p25 mice with chronic GENUS (88.56 ± 4.49) (fig. 10F, fig. 10H). Similarly, while the percentage of NeuN + nuclei (86.11 ± 2.26) in non-stimulated Tau P301S mice was significantly lower than that of the inbred wild-type littermates (100 ± 3.87), Tau P301S mice with chronic GENUS (96.05 ± 3.99) were not different from the wild-type mice (fig. 10G, fig. 10H), consistent with our immunohistochemical findings (fig. 3E, fig. 3J). These results demonstrate a reduction in neuronal nucleus loss in CK-P25 and Tau P301S neurodegenerative mouse models with chronic GENUS, and indicate an overall neuroprotective effect of GENUS.

Next, we performed RNA sequencing from these FACS sorted neuronal RNAs. On average, 18.09 and 2279 million reads were obtained per sample, with CK-P25 and P301S mice aligned from 85.23% and 84.45%, respectively. Non-deflection group analysis of NeuN + nuclei showed that there were relatively more genes down-regulated in CK-P25(618 genes) and Tau P301S (351 genes) than up-regulated genes (CK-P25: 565 genes; Tau P301S: 229 genes) compared to their respective control mice (fig. 5A, fig. 5B). Chronic GENUS resulted in a similar number of up-and down-regulated genes in CK-P25(409 up-regulation; 422 down-regulation) and Tau P301S (220 up-regulation; 221 down-regulation) compared to their respective non-stimulated controls (fig. 5A, 5B).

We then performed GO analysis to examine biological functions associated with differentially expressed genes. Neuron-specific down-regulated genes (618 genes) in CK-p25 mice after chronic GENUS were involved in chemical synaptic transmission, intracellular transport, autophagy, ATP metabolic processes, trans-synaptic signaling, and intercellular signaling (fig. 5A). Interestingly, GENUS rescued these biological processes by a significantly upregulated gene involved in these processes in CK-p25 mouse neurons (FIG. 5A; right panel). The down-regulated genes (351 genes) in TauP301S are involved in chemical synaptic transmission, trans-synaptic signaling, intracellular transport including vesicle-mediated transport, mesencephalon development, and modulation of apoptotic processes (fig. 5B). Following chronic GENUS in TAU P301S mice, these same processes-including regulation of vesicle-mediated transport, intracellular transport, synaptic transmission, mesencephalic development and apoptotic processes-were all upregulated as revealed by the most important biological functions associated with the upregulated genes (fig. 5B, and fig. 10I). On the other hand, consistent with our immunohistochemical data for γ H2AX, some biological processes in both CK-P25 and Tau P301S were upregulated (compared to the inbred controls), including those involved in DNA double strand breaks and DSB repair (fig. 9G). Importantly, genes downregulated in CK-P25 and Tau P301S mice after chronic GENUS include those known to be essential for apoptotic pathways in response to DNA damage, consistent with neuronal transition to a less degenerative state. Taken together, these data indicate that even in two neurodegenerative mouse models (CK-P25 and Tau P301S) with different mechanisms, the altered pattern of gene modulation converges to similar cellular and biological functions-including down-regulation of synaptic function, intracellular transport and apoptotic regulation-ultimately promoting neuronal death. Importantly, following chronic GENUS, many genes involved in rescuing defects in synaptic transmission, synaptic tissue, and intracellular transport including vesicle-mediated transport are upregulated (fig. 5A, 5B, and 10H).

To further characterize and validate these chronic GENUS-modified biological processes, we performed unbiased liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect differential expression of proteins and S/T phosphorylation in V1 of CK-P25 and Tau P301S mice (fig. 5C, fig. 5D). We first examined all proteins identified by mass spectrometry in the combined CK-P25 group (purebred CK control and CK-P25 with and without GENUS) and the combined Tau P301S group (wild-type purebred control and P301S with and without GENUS), respectively, and compared them to all RNAs detected from the respective neuronal specific RNA sequence data. We found that 92.75% and 91.95% of total protein was recognized in the combined CK-P25 and Tau P301S groups, respectively, mapped to expressed genes in neuron-specific RNA sequencing data (fig. 5C, fig. 5D), indicating that most of the proteins recognized are involved in neuronal function. Next, we compared the S/T phosphorylated proteins in CK-P25 and Tau P301S mice to their respective inbred control litters and found that S/T phosphorylated proteins were increased overall in CK-P25 and Tau P301S mice (FIG. 5C, FIG. 5D; bottom left panel), indicating aberrant modification of functional proteins in both neurodegenerative mouse models. Chronic GENUS resulted in decreased S/T phosphorylation of proteins in CK-P25 and Tau P301S mice compared to their respective non-stimulated controls (fig. 5C, 5D; bottom right panel). Consistent with our neuron-specific gene expression analysis (fig. 5A, 5B), the chronic GENUS-modified proteins are involved in chemical synaptic transmission, dendritic development, long-term enhancement, regulation of vesicle-mediated transport in synapses, and regulation of intracellular transport (fig. 5E and 10J to 10M), suggesting that these processes are altered in degenerated neurons and improved by chronic GENUS.

One exemplary protein related to vesicle transport, endocytosis and synaptic transmission according to our phosphoproteomics analysis is dynamin1(DNM-1) (Armbruster et al, 2013), which is associated with multiple GO terms when in differential S/T phosphoprotein annotation following chronic GENUS in CK-P25 and Tau P301S (fig. 5E). Synaptic vesicle endocytosis requires phosphorylation at Ser774 (Clayton et al, 2009) and is one of many residues that are hyperphosphorylated in CK-P25 and Tau P301S mice and reduced in chronic GENUS. We performed immunohistochemistry and western blotting and found that DMN-1Ser774 phosphorylation was significantly increased in CK-P25 and Tau P301 mice and decreased by chronic GENUS (fig. 5F and 10M, 10N). We also performed immunohistochemistry on vesicular glutamate transporter 1 (vgout 1), another protein involved in vesicular and neurotransmitter transport, synaptic transmission, and learning and memory (Balschun et al, 2009). We found that vgout 1 sites were significantly reduced in CK-p25 mice compared to naive CK mice (fig. 5G, fig. 5H), and vgout 1 expression was significantly higher in CK-p25 mice after chronic GENUS (compared to non-stimulated controls), not only in V1, but also in the ACC regions of SS1, CA1 and PFC (fig. 5G, fig. 5H). Similarly, the reduced expression of the synaptic marker vgout 1puncta was reduced in Tau P301S mice spanning these brain regions following chronic GENUS in Tau P301S mice (fig. 5I). These data are consistent with neuronal loss (fig. 3D and 3I) and synaptic loss (fig. 4O, 4P, 4Q) across these brain regions with chronic GENUS in both neurodegenerative models.

Chronic GENUS changes behavioral manifestations

To date, our findings indicate that GENUS can abolish neurooscillation (including CA1, SS1, and PFC) beyond V1 and reduce AD-associated lesions, including amyloid plaques, Tau hyperphosphorylation, synaptic loss, and neuronal loss, in all of these brain regions in CK-P25 and Tau P301S mice. Our RNA sequencing and phosphoproteomics data support the ability of chronic GENUS to alleviate synaptic transmission and intracellular transport defects associated with disease, consistent with preserved synaptic function. Therefore, we asked whether chronic GENUS also improved cognitive function. We first try to determine if GENUS triggers any systematic behavioral changes that may complicate the interpretation of any cognitive changes. In C57Bl/6J mice, there was no significant difference in locomotor activity in distance traveled during or after acute GENUS (fig. 11A-11C). Furthermore, both seizure susceptibility by tetrandrine and novel subject discrimination were unchanged (fig. 11B to 11C). Animals of all groups chronically stimulated with GENUS (C57Bl 6: 7 days; CK-p 25: 1 hour/day during p25 induction for 42 days; TauP 301S: 22 days) had body weights comparable to non-stimulated controls (FIGS. 11D-11F). Finally, seven days of GENUS did not affect the stress response marker plasma corticosterone in C57Bl/6J mice (FIG. 11I).

To assess the cognitive advantage of GENUS, we focused our studies on learning and memory in CK-p25 mouse models of AD 6 weeks after induction of p25 expression with long-term GENUS stimulation in CK-p25 mice. In the last week, mice were exposed to Open Field (OF) and then subjected to a Novel Object Recognition (NOR) test (fig. 6A). Our results show that GENUS does not affect anxiety in CK-p25 mice, as measured by the time spent in the center of the open field (fig. 6A, fig. 6B), nor does it alter plasma corticosterone levels (fig. 11I), suggesting that chronic GENUS does not affect anxiety or stress in CK-p25 mice. Interestingly, the GENUS-stimulated CK-p25 mice, while not showing any difference in locomotor activity, took significantly more time to explore novel subjects compared to familiar subjects (fig. 6A, 6C, and 11G), suggesting that chronic GENUS improved novel subject recognition in CK-p25 mice compared to non-stimulated CK-p25 mice. Next, we performed the Morris Water Maze (MWM) test on CK-p25 mice induced for 6 weeks during GENUS at week 6. In the MWM test, non-stimulated CK-p25 mice showed impaired spatial learning as shown by higher latency to find the platform during training, compared to the inbred CK mice (fig. 6D). Non-stimulated CK-p25 mice also showed impaired spatial memory as compared to the naive CK group, as less time spent in the target quadrant in the probe test (24 hours after the last training day) was visible by a reduction in the number of visits to the platform location (Fischer et al, 2005) (fig. 6D). Chronic GENUS significantly ameliorated these impairments and these impairments were not the result of changing swim speed, which remained comparable across groups and all training days (fig. 6D and 11J).

To determine that this GENUS-mediated improvement in behavioral performance is not limited to a single mechanism of cognitive impairment, but is more generally applicable to AD-related cognitive decline, we tested a number of AD model mice. We performed GENUS for 22 days (1 hour/day) on 8 month old TauP301S mice and performed OF and NOR task tests on mice on the third week OF stimulation and compared them to the age-matched non-stimulated group (fig. 6E). As with CK-P25 mice, GENUS neither altered the time spent in the center OF the OF field (fig. 6E, fig. 6F), nor the plasma corticosterone levels OF Tau P301S mice (fig. 11I), indicating that chronic GENUS did not alter the anxiety-like behavior OF Tau P301S mice. Next, we performed a novel subject identification test and observed that wild-type inbred and GENUS-stimulated Tau P301S mice exhibited a higher preference for novel subjects compared to familiar subjects (fig. 6G). Similarly, the new subjects preference was higher in the non-stimulated Tau P301S mice (fig. 6G).

Next, we performed MWM testing on Tau P301S mice at GENUS in the third week. Wild-type inbred and GENUS-stimulated Tau P301S mice showed significantly increased learning curves in MWM training compared to non-stimulated Tau P301S mice (fig. 6H and 11J). Finally, we tested the effectiveness of GENUS in improving the behavior of the AD amyloid model. During the third week of training, we performed MWM testing on GENUS-stimulated 5XFAD mice (22 days-1 hour/day) (fig. 6I). Although GENUS-stimulated 5XFAD mice exhibited a slightly lower learning curve during the first four days of MWM training, they were improved in multiple training trials compared to wild-type litters, as opposed to significantly impaired non-stimulated 5XFAD mice (fig. 6I and 11J). Finally, with 80Hz visual stimulation, we investigated whether chronic stimulation at other frequencies could alter behavior. Similar to the in vivo recordings of V1, 5XFAD mice subjected to 80Hz visual stimulation for 22 days in MWM showed no difference compared to non-stimulated 5XFAD control (quantified as the latency to look for platform and target crossover in the probe test) (fig. 12A-12C). Taken together, these results indicate that chronic stimulation at 40Hz in particular improves performance in various mouse models of neurodegeneration.

More and more evidence supports the following notions: manipulating neural network oscillations may represent a promising strategy to mitigate pathological changes and behavioral deficits associated with neurological diseases (Cho et al, 2015; Iaccarino et al, 2016; kastaneka et al, 2017; Martinez-Losa et al, 2018; Verret et al, 2012). Here, we demonstrate that entrainment of low gamma oscillations daily for a long period of time by 40Hz visual stimulation is effective in entraining gamma oscillations (even in the case of late neurodegeneration) to reduce neuropathy in multiple brain regions. The neuroprotective effects of chronic GENUS include a reduction in microglia-mediated inflammatory responses, enhanced expression of genes and proteins that promote synaptic transmission and intracellular transport in neurons, and improved performance.

Chronic vision GENUS entrains low gamma oscillations and increases gamma coherence throughout the brain region

We previously shown that in young, symptomatic, 3-month old 5XFAD mice, an acute one hour (1h) visual stimulus of 40Hz entrains neuronal activity to oscillate within the gamma frequency range and reduces the AD-associated phenotype (Iaccarino et al, 2016). In this disclosure, we demonstrate that GENUS significantly increases low gamma (-35-45 Hz) oscillating power in various parts of the brain, including the visual cortex (V1), hippocampal CA1, somatosensory cortex (SS1), and prefrontal cortex (PFC), in a CK-P25 and Tau P301S mouse model with neurodegeneration, despite severe AD-like lesions and neuronal loss. Locomotor activity may be a confounding factor in detecting gamma oscillations, although during the recording phase between occlusion and visible 40Hz visual stimuli, we detect no difference in velocity and total distance traveled, making it unlikely that the low gamma entrainment we observe during GENUS is related to differences in activity levels. Furthermore, analysis of single cell recordings in CA1 showed that GENUS was able to recruit neuronal activation in multiple brain regions downstream of V1 (see also martell and Paulson et al, along with the text) and a measurement of Weighted Phase Lag Index (WPLI), which is less sensitive to the volume conductance of uncorrelated noise sources, indicating that V1, CA1, SS1 and PFC showed enhanced low gamma coherence and thus were more functionally coupled to GENUS. Considering that communication by "Coherence Through (CTC)" is essential for cognitive function (Fries, 2015), and not surprisingly, human AD subjects have a defect in intercortical regional coherence (Stam et al, 2009).

Visual GENUS confers broad neuroprotective effects across multiple neurodegenerative mouse models

To determine whether long-term application of GENUS affects lesions throughout the brain region, we focused on other key structures of the visual cortex and default mode network, such as the hippocampus and cingulate gyrus portions of the prefrontal cortex (PFC), which have a large impact on AD. We first applied seven days of GENUS to old 5XFAD mice and then found that, as described by Iaccarino et al (2016), the amyloid levels in hippocampus were not altered despite the significant reduction of amyloid plaques in V1 (fig. 2A, 2B).

Therefore, we extended the GENUS regimen by 3 weeks using relatively older 5XFAD mice with more severe lesions (10 months of age) and found that amyloid plaque burden was significantly improved not only in V1, but also in other distributed sites of the brain including SS1, hippocampus and PFC. We applied this extended GENUS regimen in the entire 6-week P25-induced CK-P25 model and for 3 weeks in Tau P301S and 5XFAD mice at the age at which neurons began to be lost in these respective models. We found that their respective lesions, including hyperphosphorylated Tau protein, synaptic and neuronal loss and DNA damage, were significantly reduced not only to V1, but also evident in CA1, SS1 and CC (the cingulate gyrus portion of the prefrontal cortex) of the CK-P25, Tau P301S and 5XFAD mouse models. The neuroprotective effect of GENUS to prevent neuronal loss is particularly evident in the CK-p25 mouse model, which typically exhibits severe brain atrophy, cortical volume contraction and corresponding ventricular dilatation, which is associated with human AD when neurons and their expansion process degenerate. Therefore, we demonstrate that GENUS confers broad neuroprotective effects on a variety of mouse models with different pathological characteristics.

Visual GENUS modifies synaptic function, intracellular transport and behavior

To examine the effect of GENUS at the molecular level, we next turned to unbiased transcriptomic and proteomic analysis. Our data obtained from isolated/purified Tau P301S and CK-P25 neurons indicate that multiple genes, proteins, and post-translationally modified proteins that modulate synaptic function are significantly deregulated, consistent with the altered neuronal excitability and excitability/inhibitory balance previously reported in these neurodegenerative models (Fischer et al, 2005; Yoshiyama et al, 2007). AD is also known to cause a reduction in dendritic spine density, and reduction of spine density in AD mice by genetic manipulation, pharmacological inhibition of Histone Deacetylases (HDAC), or optogenetics has been demonstrated to alleviate cognitive disorders (Fischer et al, 2007); graff et al, 2012; roy et al, 2016). Furthermore, we found that chronic vision GENUS alleviated these gene expression and protein phosphorylation defects in CK-P25 and Tau P301S models, and immunohistochemical analysis with markers specific for synaptoprotein (vgout 1, basnoon) confirmed synaptic densities comparable to corresponding control mice. Changes in expression of these genes and rescue of synaptic density, as well as the enhanced low gamma coherence we see in chronic visual GENUS, suggest that chronic GENUS alters the plasticity of the visual cortex and downstream brain regions.

Post-mortem human AD samples, AD mice, iPSC models, and primary culture cells also involved disruption of intracellular transport, vesicle trafficking, and endosomal function in AD (Millecamps and Julien, 2013; Small et al, 2017; Israel et al, 2012; Cataldo et al, 2000). The link between endocytosis and A β production has been well documented by many studies (Marks and McMahon, 1998; Cirrito et al, 2008; Schobel et al, 2008; Wu and Yao, 2009). Our unbiased transcriptomic and proteomic data indicate that chronic GENUS causes alterations in gene expression and post-translational modifications at the protein level to these intracellular trafficking, vesicle trafficking, and endocytosis processes. Our findings are consistent with a reduction in increased early endosomes in CA1 in young 5XFAD mice following acute optogenetically-driven gamma oscillations (Iaccarino et al, 2016). Furthermore, our data show that chronic GENUS reduces the hyperphosphorylation of S774dynamin 1 in a mouse model of P301S and CK-P25 neurodegeneration, which affects the role of dynamin in endocytosis (Raimondi et al, 2011; Armbruster et al, 2013).

In summary, we found that enhancing neuronal survival by chronic GENUS is most likely achieved by rescuing a wide range of genes and proteins involved in modulating DNA damage responses, autophagy, synaptic transmission, intracellular transport, vesicle trafficking, and reducing neuroinflammation. In support of the broad neuroprotective effects of chronic visual GENUS to promote neuronal survival and maintain neuronal function, we found that the performance of various mouse models following chronic GENUS is improved, including improved novel subject recognition and spatial Morris water maze for CK-P25 mice and enhanced spatial water maze learning and memory for Tau P301S and 5XFAD mice. Although we observed improved performance, chronic GENUS did not alter the body weight of C57Bl/6J, CK-P25 and P301S mice (fig. 11D-11F), anxiety induced by open field exploration, or stress response measured by the marker corticosterone (fig. 11A, 11C, 11G-11I). Interestingly, Zhang et al (2015) demonstrated that chronic 2Hz visual stimulation (6 hours per day for 4 weeks) improved cognitive performance, but it did not alter a β 1-42 in the cingulate cortex of 3xTg mice.

Visual GENUS reduces neuroinflammation

The inflammatory process plays a crucial role in AD and neurodegeneration, and acute GENUS has previously been shown to cause significant morphological activation of microglia (Iaccarino et al, 2016). The specific role of microglia in neurodegeneration is complex (as it may be beneficial or detrimental), to be further studied, and recent studies have shown that the microglia phenotype can distort as the disease progresses (Mathys et al, 2017; Lee et al, 2018; ullland et al, 2017; Deczkowska et al, 2018). In this disclosure, we performed detailed morphological analysis using immunohistochemistry and microglia-specific transcriptomics analysis to more closely examine the underlying processes affected by chronic GENUS. We found that although chronic GENUS can alter microglial morphology, immune responses and catabolic processes in the CK-P25 and Tau P301S mouse models, a range of morphological phenotypes still exist.

First, Iba1 immunostaining indicated that microglia were very hyperplastic (more microglia; consistent with Mathys et al (2017)) and tended to aggregate in CK-p25 mice, which decreased after chronic GENUS. The total volume of microglia was reduced, with the exception of a subset of rod-shaped microglia, whose total somatic and primary cell volumes were higher in non-stimulated CK-p25 mice, and chronic GENUS reduced these abnormal microglia phenotypes. Interestingly, it has recently been reported that rod-like microglia are present in rat brains with diffuse brain injury, as well as in human subjects suffering from craniocerebral injury (Bachstetter et al, 2017; Taylor et al, 2014). The microglia then enter a phagocytic state to ingest a β in 5XFAD mice, which occurs in several types of GENUS (visual, auditory, or a combination of both), and morphological changes in microglia are associated with a reduction in amyloid plaque numbers (Iaccarino et al, 2016; martell and Paulson et al, ibid.). In addition, the phagocytic state of microglia is accompanied by an increase in protein degradation (GO is identified as a protein catabolic process). The inflammatory response of microglia has recently been studied in the context of neurodegeneration and AD. Our data indicate that chronic GENUS reduces the inflammatory response of microglia in CK-p25 mice, down-regulating genes (such as those involving the adaptive immune system and MHC class II) known to be up-regulated in neurodegenerative disorders (Mathys et al, 2017). Perhaps as a result of this reduction in inflammatory response, we seen a reduction in synaptic pruning marker C1q (Hong et al, 2016), while an increase in synaptic markers vgout 1 and basoon also indicates maintenance of synaptic density in chronic GENUS. It is important to note that the astrocytic response and vasculature changes following chronic auditory GENUS and a combination of auditory and visual GENUS are demonstrated in the papers attached to Martorell and Paulson et al.

In summary, our data indicate that visual GENUS can spread to multiple regions of the forebrain (SS1, CA1, PFC) and elicit responses from multiple cell types (neurons, microglia, astrocytes). Gamma entrainment using visual stimuli represents a non-invasive strategy for providing protection and improving cognitive function against neurological diseases. We found that chronic vision GENUS reduced neuronal and synaptic loss and modified genes and proteins associated with synaptic function, together supporting the behavioral improvement we observed. The present disclosure demonstrates the neuroprotective effect of chronic visual GENUS in AD mouse models to affect AD lesions in multiple brain regions and behaviors, suggesting that GENUS confers overall efficacy of neuroprotection.

Detailed description of the drawings

Fig. 1A-1J illustrate visual stimuli according to the disclosed inventive concepts entraining gamma oscillations beyond the visual cortex in multiple brain regions of a subject.

In fig. 1A, 40Hz visual stimulation (12.5 ms on and 12.5ms off) was delivered at a 50% duty cycle using the Arduino system. C57BL/6J mice were unstimulated or visually stimulated at 40Hz within 1 hour before being sacrificed and the brains stained to express C-Fos. The micro-drivers are implanted in separate groups for in vivo electrophysiological examination.

In FIG. 1B, c-Fos expression was quantified in 40 μm thick coronal brain sections to assess neuronal activity. Representative c-Fos immunostain images. The scale bar represents 50 μm. And (3) right: the 40Hz visual stimulation significantly increased the neuronal activity marker c-Fos in the visual cortex (V1; N ═ 4 mice/group, independent sample T test, T ═ -7.110, P ═ 0.002), somatosensory cortex (SS 1; T ═ -5.239, P ═ 0.006), hippocampus (CA 1; T ═ -4.989, P ═ 0.008) and prefrontal cortex (CC; T ═ 2.938, P ═ 0.01) compared to non-stimulated mice.

In FIG. 1C, the power spectrum of the C57BL/6J mouse LFP from V1, SS1, CA1, and PFC is recorded. Red lines indicate recording during visual 40Hz visual stimulus presentation, while blue lines indicate blocked 40Hz lamp blinking (cover LED array to block light; see methods; N ═ 7 mice).

In FIG. 1D, the group gamma area power (40. + -. 5Hz) was calculated from FIG. 1C. The 40Hz visual stimulus significantly increased across V1(Wilcoxon-Ranksum, Z5.9, P3.1 x 10) compared to control conditions (LED flicker occluded)^-9)、SS1(Z＝2.4,P＝0.018)、CA1(Z＝3.4,P＝6.9x 10^-4) And PFC (Z ═ 3.3, P ═ 9.2x 10^-4) The gamma power of (d).

In fig. 1E, we implanted custom quadrupoles microdrive into C57BL/6J mice, adjusted the quadrupoles to CA1 and isolated the individual units. The graph shows the probability of spikes across the 40Hz phase within the light-blocking and 40Hz stimulation periods.

In fig. 1F, 40Hz visual stimulation was shown to significantly increase the lock-in intensity of the pyramidal neuron spikes to local LFP as compared to light block conditions (oc. led), as analyzed by averaging the resulting lengths (MRL) (24 cells of 4 mice N. Wilcoxon-Ranksum, 2.5Z, 0.011P).

In fig. 1G, LFP coherence between structures was quantified using the weighted phase lag exponential method (WPLI; N ═ 7 mice), indicating the recording sites.

In FIG. 1H, the group variation in the low gamma band (30-50Hz) WPLI associated with FIG. 1G is shown. The 40Hz visual stimulus results in a significant increase in coherence between V1-CA1(Wilcoxon-Ranksum, Z ═ 2.2, P ═ 0.03), V1-SS1(P ═ 0.021) and V1-PFC (Z ═ 2.5, P ═ 0.014).

Fig. 1I is a schematic diagram of delivering an 80Hz LED lamp at a 50% duty cycle (6.25 ms on and 6.25ms off).

Fig. 1J, to the left, shows the power spectrum of V1LFP in C57Bl/6J mice subjected to light blocking (oc. led) or 80Hz visual stimulation. For the right side, the area power centered at 80Hz (± 5Hz) was not significantly different from the 80Hz visual stimulus (N ═ 5 mice, test Wilcoxon-Ranksum, Z ═ 1.2, P ═ 0.22).

Fig. 2A-2F show that chronic 40Hz (but not 80Hz) visual flicker stimulation according to the disclosed inventive concepts reduces amyloid plaques beyond the visual cortex in a subject.

Figure 2A shows amyloid plaque burden in 5XFAD mice exposed to 1 hour/day, 7 consecutive days without stimulation, 40Hz or 80Hz visual stimuli, as seen by immunohistochemical staining of the D54D2 antibody. Representative images from V1, SS1, and CA1 in each case, scale bar represents 50 μm.

Figure 2B shows a panel quantification showing that GENUS reduces amyloid plaques in V1 1 hour per day for 7 days, but it does not significantly alter amyloid plaques in SS1 or hippocampal CA 1. Visual stimulus exposure at 80Hz did not alter amyloid plaque levels in V1 or CA1, but significantly increased amyloid plaques in SS 1. N-8 non-stimulated and 6 mice, each from the 40Hz and 80Hz groups. The interclass two-way anova effect F (2,51) ═ 5.378, and P ═ 0.0076. Post multiple comparisons of Bonferroni, P <0.001, P < 0.05.

Figure 2C shows a representative image of amyloid plaque burden in 5XFAD mice exposed to the extended GENUS regimen without stimulation or for 22 days (1 hour per day). The scale bar represents 50 μm.

Figure 2D shows that 22 days of GENUS significantly reduced amyloid plaques in V1, SS1, CA1, and CC. N ═ 6 mice per condition. Interclass two-way anova effect F (1,40) ═ 51.00, P < 0.0001. Multiple comparative tests of Bonferroni, P <0.001, P ≦ 0.01.

Figure 2E shows representative images of amyloid plaques in V1, SS1, and CA1, as observed from mice exposed to no stimulation or 80Hz lamp flashes, 1 hour per day, for 22 days, with a scale bar of 50 μm.

Fig. 2F is related to fig. 2E and shows the group data quantifying amyloid plaques in V1, SS1, and CA 1. N-6 mice per group. Two-way anova measure F (1,30) was found to be 0.0033 and P0.9565 to have no significant difference between groups.

Fig. 3A-3J show that chronic visual stimulation according to the disclosed inventive concepts improves the pathology associated with alzheimer's disease and significantly reduces or prevents neurodegeneration in a subject.

Figure 3A provides an experimental summary showing that P301S mice were subjected to no stimulation or GENUS for 22 days (1 hour per day), followed by immunohistochemistry and phosphoproteomics analysis. Wild type cage litters that did not undergo a stimulation protocol were considered wild type inbred mice.

FIG. 3B provides representative images showing S202/T205 phosphorylated tau immunostaining from the visual cortex. Scale bar 50 μm. And (3) right: P301S tau mice showed higher levels of S202/T205 in V1, SS1, CA1 and CC, while 22-day GENUS showed significantly reduced levels of S202/T205 in all regions examined. N-7 wild-type inbred, 8 unstimulated P301S mice and 7 GENUS-stimulated P301S mice. Interclass two-way anova effect F (2,76) ═ 45.35, P < 0.0001. Post multiple comparisons were performed with Bonferroni's correction, P <0.0001, P <0.001, P <0.01, P < 0.05.

Figure 3C shows a phosphoproteomic analysis of serine/threonine phosphorylated tau proteins from the visual cortex. The heat map shows S/T residues of differentially phosphorylated tau protein in P301S compared to wild type mice. Interclass two-way anova effect F (2,322) ═ 2146, P < 0.0001. All phosphopeptides were plotted as described in the methods. All S/T residues of tau shown in the heatmap were statistically significant between wild-type inbred and unstimulated P301S. Statistical comparisons of specific residues (P-values) between non-stimulated and GENUS-stimulated P301S mice are shown in the graph. GENUS reduced phosphorylation of tau at 6S/T sites and increased phosphorylation of S451. The common human tau S/T site is shown at the top.

Fig. 3D shows representative images of the neuronal marker NeuN in the visual cortex from wild type inbred and P301S mice that were subjected to no stimulation or GENUS. The scale bar represents 50 μm.

Figure 3E shows panel data showing that P301S mice showed significant loss of neurons in V1, SS1, CA1 and CC compared to wild type inbred mice. The GENUS stimulated P301S group showed significantly reduced neurodegeneration. N is the same as in fig. 3B. Interclass two-way anova effect F (2,76) ═ 19.73, P < 0.0001. Multiple comparisons post hoc were performed with Bonferroni's correction, # P <0.001, # P <0.01, # P < 0.05.

FIG. 3F provides an experimental summary showing the induction of p25 expression in CK-p25 mice for 42 days. In one experimental group, with GENUS, 1 hour per day, the non-stimulated control group received room illumination. CK (CaMK2 alpha-promoter x tTA) cage litters that did not undergo a stimulation protocol were considered as inbred CK mice.

FIG. 3G is a micrograph showing qualitative differences in brain between inbred CK, non-stimulated and GENUS-stimulated CK-p25 mice 42 days post-induction. And (3) right: CK-p25 mice exhibited reduced brain weight (i.e., brain atrophy) compared to naive CK mice, while chronic GENUS partially alleviated brain atrophy in CK-p25 mice. N-13 pure CK mice, 10 mice each in the non-stimulated and GENUS CK-p25 groups. Analysis of variance F (2,30) ═ 15.46, P < 0.001. Multiple comparisons post hoc were performed with Bonferroni's correction, P <0.0001, P < 0.05.

Fig. 3H provides a representative image and size quantification (overview) of the lateral ventricles, scaled to 1000 μm. And (3) right: CK-p25 mice exhibited abnormal ventricular dilatation, which was significantly reduced after chronic GENUS, compared to the naive CK mice. N-9 pure CK mice and 6 mice from each of the non-stimulated and GENUS CK-p25 groups. Analysis of variance F (2,18) 12.36 with P < 0.001. Multiple comparisons post hoc were performed with Bonferroni's correction, P <0.0001, P < 0.05. See also fig. 9E.

Figure 3I provides representative images of the neuronal marker NeuN in the visual cortex from purebred CK and CK-p25 mice that were subjected to no stimulation or GENUS. The scale bar represents 50 μm.

FIG. 3J shows that CK-p25 mice showed severe neuronal loss in V1, SS1, CA1 and CC, whereas chronic GENUS reduced neuronal loss in CK-p25 mice. N is the same as in fig. 3G. Bidirectional anova F (2,72) ═ 31.38, P < 0.0001. Multiple comparisons post hoc were performed with Bonferroni's correction, P <0.001, P <0.01, P < 0.05.

Fig. 4A-4Q show that chronic visual stimuli according to the disclosed inventive concepts reduce the inflammatory response of microglia in a subject.

Figure 4A relates to CK-p25 mice that were subjected to no irritation or GENUS for 1 hour per day for 42 days. Microglia were then isolated from the visual cortex using fluorescence activated cell sorting (FACS; using dual positive criteria of CD11b and CD 45), followed by RNA extraction and sequencing. Differentially Expressed Genes (DEG) are shown in the volcano plots. Group comparisons are shown at the bottom. Left: DEG between pure CK and unstimulated CK-p25 mice, N ═ 4 mice per group. And (3) right: DEG between non-stimulated and GENUS CK-p25 mice, N ═ 4 mice per group.

Figure 4B shows selected Gene Ontology (GO) terms for biological processes associated with the identified DEG. Top: in contrast to the inbred CK mice, GO terminology was associated with UP-regulated (UP) genes in non-stimulated CK-p25 mice. Bottom: in contrast to the inbred CK mice, GO terminology was associated with DOWN-regulated (DOWN) genes in non-stimulated CK-p25 mice.

FIG. 4C shows selected GO terms associated with a DEG. Top: GO terms are associated with UP-regulated (UP) genes in GENUS CK-p25 mice compared to non-stimulated CK-p25 mice. Bottom: GO terms are associated with DOWN-regulated (DOWN) genes in GENUS CK-p25 mice compared to non-stimulated CK-p25 mice.

Figure 4D provides representative images showing immunostaining of the microglia markers Iba1 (green) and CD40 (red) from the visual cortex. Scale bar 50 μm. N-7 pure CK mice, 6 mice each in the non-stimulated and GENUS groups. Arrows and arrows indicate the branching volumes of rod-like branching processes and microglial processes, respectively.

FIG. 4E shows that non-stimulated CK-p25 mice exhibited higher numbers of Iba1 positive cells compared to pure CK mice, and chronic GENUS significantly reduced Iba1 cell density in CK-p25 mice. Analysis of variance F (2,16) ═ 17.79, P < 0.0001.

In fig. 4F, we performed three-dimensional rendering of microglia using Imaris and quantified the volume of somatic cells and the process of microglia. N-microglia per group from the same number of mice as in fig. 4D. Frequency distribution of the volume (size) of microglial cells that did not show any statistical significance between groups (Kruskal-Wallis test, H-0.3529, P-0.8382).

Figure 4G shows that the total volume of microglial processes (excluding rod-shaped microglia) was significantly lower in non-stimulated CK-p25 mice than in pure CK mice. GENUS-stimulated CK-P25 mice showed no difference compared to the naive CK mice (H9.224, P0.009).

Fig. 4H shows quantification of the minimum distance between microglia. N-68 microglia from 9 pure CK, 131 microglia from 6 non-stimulated mice and 95 microglia from 6 GENUS CK-p25 mice. Microglia aggregated together in non-stimulated CK-P25 mice compared to pure CK mice, whereas GENUS significantly reduced microglia aggregation (H ═ 100.1, P < 0.0001).

4I shows the quantification of the radial primary process of rod-shaped microglia. N-16 microglia from 9 pure CK, 19 microglia per group from 6 mice, each from non-stimulated and GENUS CK-p25 groups. The total volume of rod-shaped microglial processes was significantly reduced in CK-p25 mice after GENUS compared to non-stimulated CK-p25 mice. Analysis of variance F (2,51) ═ 16.27, P < 0.0001.

Figure 4J shows that non-stimulated CK-p25 mice exhibited higher signal intensity of interferon-responsive protein CD40 compared to pure CK mice, while chronic GENUS significantly reduced CD40 signal in CK-p25 mice. Analysis of variance F (2,16) ═ 36.84, P < 0.0001.

FIG. 4K provides a representative image from the visual cortex showing that Iba1 is green and the nucleus is stained Hoechst blue. Scale bar 50 μm. Arrows indicate the complexity of the microglial process.

FIG. 4L shows that P301S tau mice showed a trend towards an increased total number of Iba1 positive cells, but were not statistically significant compared to wild type mice. N-7 wild-type inbred, 8 unstimulated P301S and 7 GENUS mice. Analysis of variance F (2,19) ═ 2.401, and P ═ 0.1190.

Figure 4M is a histogram showing the size of small glial cells. N-7 wild-type inbred, 8 unstimulated P301S and 7 GENUS mice. A total of 58 microglia were analyzed per group. There was no difference in the total volume of microglial cells between groups. Kruskal Wallis test H-0.04269 and P-0.9789.

Figure 4N shows that the volume of microglial processes was smaller in P301S tau mice compared to wild-type mice, while GENUS-stimulated P301S mice showed similar process length as wild-type mice. Kruskal Wallis test H-7.895 and P-0.0193.

Fig. 4O provides a representative image showing immunostaining of C1q (a classical complement pathway mimicking protein) from the visual cortex. Scale bar 50 μm. Top: n-7 pure CK mice, 6 mice were each non-stimulated and GENUS-stimulated groups. Bottom: n-7 wild-type inbred, 8 unstimulated P301S and 7 GENUS mice.

FIG. 4P shows that non-stimulated CK-P25 mice exhibited higher C1q signal intensity, while chronic GENUS-stimulated CK-P25 mice exhibited significantly reduced C1q intensity, as compared to pure CK mice. Analysis of variance F (2,16) ═ 13.39, P ═ 0.0004.

Figure 4Q shows that non-stimulated P301S mice exhibited higher C1Q signal intensity compared to wild type inbred mice, with no difference in chronic GENUS between any of the groups. Analysis of variance F (2,19) ═ 6.887, P ═ 0.005. Multiple comparisons post hoc at E, I, J, L, P were performed with Bonferroni's correction, Q × P <0.0001,. P <0.05, n.s-not significant.

Fig. 5A-5I illustrate that chronic visual stimuli according to the disclosed inventive concepts alter synaptic function and intracellular transport in neurons.

In FIG. 5A, CK-p25 mice were subjected to no irritation or GENUS for 42 days, 1 hour per day. NeuN-positive neuronal nuclei (100,000) were isolated from the visual cortex using FACS, followed by RNA extraction and sequencing. Heat map of DEG. The number of mice per group is indicated in the heatmap. Upper left: DEG between naive CK and non-stimulated CK-p25 mice. Left lower: DEG between non-stimulated and GENUS-stimulated CK-p25 mice, gene numbers are indicated on the right. And (3) right: the graph shows the overlap of biological processes associated with down-regulated genes in unstimulated CK-p25 mice and the same biological processes associated with up-regulated genes in CK-p25 mice after GENUS, compared to inbred CK mice.

In fig. 5B, P301S mice were subjected to no stimulation or GENUS stimulation for 1 hour per day for 22 days. NeuN-positive neuronal nuclei (100,000) were isolated from the visual cortex using FACS, followed by RNA extraction and sequencing. Heatmap of differentially expressed genes. The number of mice per group is indicated in the heatmap. Upper left: DEG between wild type inbred and non-stimulated P301S tau mice. Left lower: DEG between non-stimulated and GENUS-stimulated P301S tau mice, gene numbers indicated on the right. And (3) right: the graph shows the overlap of biological processes associated with down-regulated genes in non-stimulated P301S mice compared to pure CK mice and the same biological processes associated with up-regulated genes in P301S mice after GENUS stimulation.

In FIG. 5C, CK-p25 mice were subjected to no irritation or GENUS for 42 days. Total protein expression and S/T phosphorylated protein analysis were performed on visual cortical tissue using TMT 10-plex kit and mass spectrometry. N-3 pure CK, 3 unstimulated CK-p25 and 4 GENUS-stimulated CK-p25 mice. Top: the venn plot shows the overlap of total RNA identified from the neuron-specific RNA sequence (fig. 5B above) and total protein identified from LC-MS/MS. 92.73% of the identified protein was found to be expressed in neurons. Bottom: volcano patterns of differential S/T phosphorylated protein in CK-p25 mice compared to control littermates (left) and GENUS (right).

In fig. 5D, P301S tau mice were subjected to no stimulation or GENUS stimulation for 22 days. Total protein expression and S/T phosphorylated protein analysis were performed on visual cortex tissues using a tandem mass spectrometry tag (TMT)10-plex kit (see methods) and mass spectrometry (LC-MS/MS). N-3 wild-type inbred, 3 unstimulated P301S and 4 GENUS-stimulated P301S mice. Top: the venn plot shows the overlap of total RNA identified from the neuron-specific RNA sequence (fig. 5A above) and total protein identified from LC-MS/MS. The identified 91.95% of the protein was found to be expressed in neurons. Bottom: volcano patterns of differentiated S/T phosphorylated proteins in P301S tau mice compared to control littermates (left) and GENUS (right). Phosphorylated proteins with a fold change of ± 0.2 and an adjusted P-value <0.05 were considered statistically significant.

FIG. 5E shows GO terminology for differentiated S/T phosphorylated proteins in CK-P25 and P301Stau mice after chronic GENUS.

Fig. 5F shows representative images showing immunostaining of neurofilament heavy chain (NFH, a neuronal marker expressed primarily in axons) and dynamin1 phosphorylated at Ser774 from the visual cortex. NFH is used to label neuronal processes. Scale bar 10 μm. N-7 pure CK mice, 6 mice were each non-stimulated and GENUS-stimulated groups. The middle part: CK-p25 mice exhibited significantly higher levels of pS774-DNM1 signal intensity compared to pure CK, while GENUS significantly reduced this aberrant phosphorylation of CK-p25 mice. F (2,16) ═ 38.551, P < 0.0001. Multiple comparative tests of Bonferroni, P <0.001, P < 0.01. And (3) right: non-stimulated P301S tau mice exhibited significantly higher levels of pS774-DNM1 signal intensity compared to wild-type inbred mice, while GENUS significantly reduced this abnormal phosphorylation in P301S mice. N-7 wild-type inbred, 8 unstimulated P301S and 7 GENUS mice. Analysis of variance F (2,19) ═ 18.69, P < 0.0001. Multiple comparisons post hoc were performed with Bonferroni's correction, # P <0.01, n.s-not significant.

FIG. 5G shows representative brain sections immunostained for vesicular glutamate transporter 1(vGlut1) from the visual cortex of inbred CK, unstimulated and GENUS-stimulated CK-p25 mice. The scale bar represents 10 μm.

FIG. 5H shows that, although the expression of vGlut1 point was significantly reduced in non-stimulated CK-p25 mice, GENUS-stimulated CK-p25 mice exhibited higher levels of vGlut1 point in V1, SS1, CA1, CC than in non-stimulated CK-p25 mice. N-9 pure CK mice, and 6 mice each from the non-stimulated and GENUS CK-p25 groups. Interclass two-way anova effect F (2,72) ═ 42.06, P < 0.0001. Multiple comparisons post hoc were performed with Bonferroni's correction, P <0.0001, P <0.01, P < 0.05.

Fig. 5I shows that the expression of the synaptic point vgout 1 in P301S was significantly reduced compared to wild-type inbred mice, and GENUS rescued the expression of vgout 1 in V1, CA1 and CC of P301S. N-7 wild-type inbred, and 8 non-stimulated P301S and 7 GENUS mice. Interclass two-way anova effect F (2,76) ═ 23.67, P < 0.0001. Post multiple comparisons were performed with Bonferroni's correction, P <0.0001, P <0.001, P <0.01, P < 0.05.

Figures 6A-6I illustrate that chronic visual stimuli according to the disclosed inventive concepts alter behavior in multiple subject models of alzheimer's disease.

In fig. 6A, p25 expression was induced in two groups of CK-p25 mice for 42 days, of which only one group received 1 hour of daily GENUS (stimulation between 9 am and 12 pm). Open Field (OF) and novel object identification (OR) tests were performed on mice on day 40 (OF) and day 41 (OR) afternoon (3 pm to 7 pm). F and N represent familiar objects and novel objects, respectively. Representative occupancy heatmaps from the OF and OR phases are shown. Color levels map to a range of positional frequencies in the stage or object: warm colors indicate higher times and frequencies in a given location, while cool colors indicate less time.

FIG. 6B shows that GENUS does not affect anxiety levels in CK-p25 mice. There was no difference in the time spent in the OF stage center between inbred CK, non-stimulated and GENUS-stimulated CK-p25 mice. Analysis of variance effect between groups F (2,49) ═ 1.198 and P ═ 0.3104.

However, fig. 6C shows that the pure CK and GENUS stimulated CK-p25 mice had a significantly higher preference for novel subjects than familiar subjects; non-stimulated CK-p25 mice showed no preference. The two-way analysis of variance effect between the familiar subject and the novel subject, F (1,70) ═ 7.742, P ═ 0.0069. T inspection; pure CK, T-4.421, P-0.0001; CK-P25+ is non-irritant, T is 1.108, P is 0.0946; CK-P25+ GENUS, T3.306, P0.0017.

FIG. 6D shows the Morris water maze performance of p 25-induced CK-p25 mice with and without 42 days of GENUS (MWM; between day 36 and day 41 of p25 induction). Top: chronic GENUS reduced the latency to find the platform in CK-p25 mouse training compared to non-stimulated CK-p25 mice. Interclass two-way anova effect F (2,252) ═ 18.64, P < 0.0001. Bottom: the number of platform crossings in the probe test performed 24 hours after the final training phase was significantly higher in the GENUS stimulated group compared to non-stimulated CK-P25 mice (left: interclass effect F (2,42) ═ 5.277, P ═ 0.0090). Compared to the naive CK mice, the time spent by non-stimulated CK-P25 mice in the target quadrant (right; effect between groups F (2,42) ═ 6.35, P ═ 0.0039) was significantly lower.

Fig. 6E refers to P301S tau mice exposed to no irritation OR GENUS for 1 hour per day for 22 days (9 am to 12 pm) and then evaluated in the OF (day 20; 3 pm to 7 pm) and OR (day 21) tests. Representative occupancy heatmaps from the OF and OR phases are shown.

Figure 6F shows that GENUS did not affect anxiety levels in P301S mice. There was no difference in the time spent in the OF centers for non-stimulated, GENUS-stimulated P301S and wild-type pure cohorts (anova F (2,36) ═ 1.189, P ═ 0.3163).

Figure 6G shows that wild-type, non-stimulated and GENUS-stimulated P301S mice all showed higher preference for novel subjects. The two-way analysis of variance effect between the familiar subject and the novel subject F (1,80) is 88.61, P < 0.0001. T inspection; wild type pure breed, T is 6.525, P < 0.00001; P301S + is non-irritating, T is 2.602, P is 0.0054; P301S + GENUS, T10.65, P < 0.00001.

Figure 6H shows MWM performance of wild type inbred, unstimulated or GENUS-stimulated P301S tau mice. Top: chronic GENUS reduced the latency to find the platform during training of P301S mice compared to non-stimulated P301S mice. The inter-group two-way anova effect F (2,225) ═ 3.782, P ═ 0.0242. Bottom: the number of platen crossings in the probe test (left: F (2,45) ═ 2.872, P ═ 0.067) and the time in the target quadrant in the probe test (right: F (2,45) ═ 3.115, P ═ 0.054.

In fig. 6I, 5XFAD mice were tested for MWM performance with or without GENUS (1 hour per day for 22 days). Left: the latency of looking for a platform during training. Interclass two-way anova effect F (2,273) ═ 16.97, P < 0.0001. In non-stimulated CK-P25 mice, the number of platform crossings in the probe test (middle: interclass effect F (2,39) ═ 4.702, P ═ 0.0148) and the time spent in the target quadrant (right: interclass effect F (2,39) ═ 7.289, P ═ 0.0020) were significantly lower.

Fig. 7A-7I show that chronic visual stimulation according to the disclosed inventive concepts entrains gamma oscillations beyond the visual cortex in a mouse model of neurodegeneration.

Fig. 7A shows electrolytic lesions to verify the recording sites in mice recorded after fig. 1D, used in main plot 1C. Representative images show the recording sites for V1, SS1, CA1, and PFC.

As shown in fig. 7B, CDK5 activator p25 was induced in CK-p25 mice for 6 weeks, followed by microdrive implantation and LFP recording.

In fig. 7C, the regional power (35-45Hz) in V1, CA1 and PFC was significantly lower for CK-P25 mice compared to age-matched wild-type mice (all regions P < 0.01). During 40Hz lamp flash exposure, 40Hz visual stimuli increased 40Hz power in V1(Wilcoxon-Rank sum; P0.0022), CA1 (P0.001) and PFC (P0.002) in 6-week-induced CK-P25 mice.

In fig. 7D, the microactuator was implanted in an 8-month old P301S tau mouse.

Fig. 7E shows that a significant increase in power (35-45Hz) was observed during 40Hz light stimulation in V1(P ═ 6.01E-05) and PFC (P ═ 4.11E-05).

FIG. 7F relates to C57Bl6/J mice that were subjected to no irritation or GENUS for 1 hour/day for 42 days. And (3) right: a significant increase in area power of 35-45Hz was observed during 40Hz visual stimulation in V1(Wilcoxon-Ranksum test; P ═ 1.54E-06), SS1(P ═ 2.88E-04), CA1(P ═ 0.04), and PFC (P ═ 3.39E-06).

In FIG. 7G, C57Bl6/J mice were subjected to no stimulation or GENUS for 1 hour/day for 42 days. Mice were visually stimulated at 40Hz on day 43 and LFP was collected. During GENUS, a significant increase between regions was observed in 30-50Hz low gamma coherence (measured as weighted phase lag index) between V1-CA1(P ═ 0.002), V1-SS1(P ═ 0.008), CA1-PFC (P ═ 0.04), and V1-PFC (P ═ 0.002). There was no difference in low γ WPLI between CA1 and SS1(P ═ 0.18).

In FIG. 7H, p25 was induced in CK-p25 mice for 6 weeks. Six weeks induced CK-p25 mice were either non-stimulated or GENUS (from day 43 onwards), 1 hour/day, for 42 days. And (3) right: in all brain regions studied in CK-P25 mice, a significant increase in low gamma area power (35-45Hz) was observed [ V1, P ═ 0.0017), CA1(P ═ 0.0379), PFC (P ═ 0.0030) ] (measured on day 85).

Fig. 7I shows that low gamma coherence between V1-CA1(P <0.01), CA1-PFC (P <0.01), V1-PFC (P <0.01) is also significantly increased.

Fig. 8A-8I show that chronic visual stimuli according to the disclosed inventive concepts reduce AD-associated lesions beyond the visual cortex in 5XFAD mice.

Fig. 8A is a schematic diagram illustrating a visual flicker stimulation apparatus. An array of Light Emitting Diodes (LEDs) was present on the open side of the cage and was driven to flash at a frequency of 40Hz using the Arduino system in a square wave current mode. Since the mice were free to move within the cages (total area 83 square inches), they received light at intensities of-1500 to 300 lux.

In fig. 8B, a 10 month old 5XFAD mouse was subjected to no stimulation or GENUS for 1 hour per day for 22 days. Representative images show red amyloid plaques and blue nuclear staining Hoechst from sections containing hippocampus and somatosensory cortex. Scale bar 1000 μm. This is related to FIGS. 2C to 2D.

In fig. 8C, a 10 month old 5XFAD mouse was subjected to no stimulation or GENUS for 1 hour per day for 22 days. Representative images show red amyloid plaques, the green neuronal marker NeuN, and blue nuclear staining Hoechst from the cingulate cortex. Scale bar 50 μm. This is related to FIGS. 2C to 2D.

Figure 8D shows that the number of plaques in GENUS-stimulated 5XFAD mice was significantly lower than non-stimulated 5XFAD mice in V1, SS1, CA1, and CC. This is related to FIGS. 2C to 2D. N ═ 6 mice per condition. Interclass two-way anova effect F (1,40) ═ 30.01, P < 0.0001. P <0.001, P <0.01, P < 0.05.

In fig. 8E, significant reduction in neuronal density in CA1 and CC was shown in 9 month old unstimulated 5XFAD mice compared to age-matched wild-type litters. GENUS significantly reduced neuronal loss in CC of 5XFAD mice. N-9 wild-type pure species, 6 mice with 5XFAD + without stimulation and 6 mice with 5XFAD + GENUS. Interclass two-way anova effect F (2,72) ═ 14.93, P < 0.0001. Multiple comparative tests of Bonferroni P <0.001, P <0.01, P < 0.05.

In fig. 8F, a 10 month old 5XFAD mouse was subjected to no stimulation or GENUS for 1 hour per day for 22 days. Representative images show the synaptic marker, bassoon. Scale bar 10 μm.

In fig. 8G, 5XFAD mice showed a significant reduction in synaptic marker, basoon staining in CA1 and CC, which was significantly reduced at 40Hz GENUS. N-9 wild-type pure species, 6 mice with 5XFAD + without stimulation and 6 mice with 5XFAD + GENUS. Interclass two-way anova effect F (2,72) ═ 16.18, P < 0.0001. Multiple comparative tests of Bonferroni P <0.001, P <0.01, P < 0.05.

Fig. 8H provides a full-length immunoblot showing expression levels of full-length APP protein and an endogenous GAPDH control. N-5 wild-type inbred mice, 3 unstimulated 5XFAD mice and 4 GENUS-stimulated 5XFAD mice.

Figure 8I shows that APP protein expression is significantly higher in 5XFAD mice compared to wild type inbred controls. There was no difference between non-stimulated and GENUS-stimulated 5XFAD mice. Note that we did not measure the C/N fragment of the APP protein. C-terminal specific APP antibody, one-way anova F (2,9) ═ 4.436, P ═ 0.046. N-terminal specific APP antibody, one-way anova F (2,9) ═ 13.194, P ═ 0.002. Multiple comparative tests by Bonferroni, # P < 0.01.

Fig. 9A-9G show that chronic visual stimulation according to the disclosed inventive concept improves AD-associated lesions in P301S and CK-P25 mice.

Figure 9A provides a full-length immunoblot showing expression levels of total tau protein and GAPDH from the visual cortex of non-stimulated, GENUS-stimulated P301S tau mice and age-matched wild-type inbred litters. And (3) right: however, total tau expression was increased in P301S tau mice compared to wild type inbred mice; there was no difference in tau levels between non-stimulated and GENUS-stimulated P301S mice. N-3 wild-type inbred, 3 unstimulated and 4 GENUS-stimulated P301S mice. Analysis of variance F (2,7) ═ 173.275, P < 0.0001. Multiple comparative tests by Bonferroni P < 0.001.

FIG. 9B shows no difference in brain weight among wild type inbred, unstimulated P301S and GENUS stimulated P301S mice. There was a tendency for lateral ventricle abnormal expansion in P301S tau mice compared to wild type inbred mice. Analysis of variance F (2,19) ═ 2.761, P ═ 0.079.

Figure 9C provides a full-length immunoblot showing expression levels of p25+ GFP (fusion protein), p25, p35, and GAPDH from the visual cortex of inbred CK, non-stimulated and GENUS-stimulated CK-p25 mice. And (3) right: however, there was a significant increase in P25+ GFP protein in CK-P25 mice compared to age-matched naive CK mice (one-way anova, F (2,12) ═ 13.065, P ═ 0.002); there was no difference in p25+ GFP expression between non-stimulated and GENUS-stimulated CK-p 25. Similarly, the expression level of P25 was significantly higher in CK-P25 mice than in the pure CK mice, but there was no difference between non-stimulated and GENUS-stimulated CK-P25 mice (one-way anova, F (2,12) ═ 3.581, P ═ 0.025). Multiple comparative tests of Bonferroni P <0.001, P <0.01, P < 0.05.

FIG. 9D provides representative images showing the thickness of the visual cortex in mice of inbred CK, non-stimulated CK-p25, and GENUS-stimulated CK-p 25. Nuclear staining Hoechst is shown in blue and the neuronal marker NeuN is shown in red. And (3) right: the bar graph shows the difference in visual cortex thickness between groups. Interclass two-way anova effect F (2,36) ═ 12.93, P < 0.0001. Multiple comparative tests of Bonferroni P <0.001, P <0.01, P < 0.05.

Fig. 9E provides representative images showing qualitative differences between inbred CK, non-stimulated and GENUS-stimulated CK-p25 mice, detailing changes in hippocampal volume, cortical thickness, and ventricle size. This is related to FIGS. 3F to 3H.

FIG. 9F shows that GENUS did not alter the P25+ GFP fusion protein expression level (P25 numbers: GFP expressing neurons) compared to non-stimulated CK-P25 mice, with P >0.05 in all brain regions tested, independent samples from groups t-test.

Fig. 9G provides representative images showing the expression of the DNA double strand break marker γ H2Ax from CA1 in inbred CK, non-stimulated and GENUS-stimulated CK-p25 mice. There were no significant γ H2Ax positive nuclei in the inbred CK mice, and therefore comparisons were made between non-stimulated and GENUS-stimulated CK-p25 mice. N ═ 6 mice per condition. Scale bar 100 μm. And (3) right: GENUS significantly reduced γ H2Ax positive nuclei in V1 (independent sample T test; T4.418, P0.0006), SS1 (T2.944, P0.013), CA1 (T2.664, P0.0143) and CC (T1.883, P0.055).

Fig. 10A-10N show that chronic visual stimulation according to the disclosed inventive concepts alters microglia, improving intracellular transport and synaptic transmission in neurons.

FIG. 10A shows microglial separation profiles in FACS from representative inbred CK, unstimulated and GENUS-stimulated CK-p25 mice. This is related to the main pattern 4A and FIG. 4C.

Figure 10B shows microglial separation profiles in FACS from representative wild type inbred, non-stimulated and GENUS-stimulated P301S tau mice.

In fig. 10C, Differentially Expressed Genes (DEG) are shown in the volcano plot. Group comparisons are shown on the right. Top: DEG between wild type inbred and unstimulated P301S, N — 5 mice per group. Middle upper part: DEG between non-and GENUS-stimulated P301S mice, N — 5 mice per group.

Figure 10D shows the top 7 processed Gene Ontology (GO) terms for biological processes associated with the identified DEG. Group comparisons are shown at the top.

Fig. 10E provides representative images showing Iba1 in green and CD40 in red. The merged image presented in main fig. 4D is isolated for clarity.

FIG. 10F shows neuronal nuclear segregation profiles in FACS from representative inbred, unstimulated and GENUS-stimulated CK-p25 mice. Group mean and SEM of% NeuN compared to total nuclei: pure CK, 50 +/-1.48; non-stimulatory CK-p25, 40.98 ± 0.719; GENUS-stimulated CK-p25 mice, 45.08. + -. 1.691. Analysis of variance F (2,12) ═ 11.55, P ═ 0.0016. In the post-test of Bonferroni, pure CK Vs non-stimulated CK-P25, P0.001', pure CK Vs GENUS stimulated CK-P25, P0.0609. GENUS reduced neuronal nuclear loss in CK-p25 mice.

Figure 10G shows neuronal nuclear segregation profiles in FACS from representative wild type inbred, unstimulated P301S and GENUS stimulated P301S mice. Group mean and Standard Error of Mean (SEM) of percent NeuN positive nuclei found compared to total Hoechst positive nuclei: wild type pure breed, 52.45 + -2.03; non-stimulated P301S, 45.17 ± 1.18; GENUS stimulated P301S, 50.38 + -2.09. Analysis of variance F (2,18) ═ 4.97, P ═ 0.0191. Post hoc testing by Bonferroni, wild type inbred Vs non-stimulated P301S, P ═ 0.028; wild type inbred Vs GENUS stimulated P301S mouse, P ═ 0.99. GENUS reduced neuronal nuclear loss in P301S tau mice.

Fig. 10H provides a bar graph showing the percentage of total neuron (NeuN) positive nuclei compared to total nuclei, as shown in fig. 10F and 10G. Top: the bar graph compares wild-type inbred, unstimulated and GENUS-stimulated P301S mice. N-7 mice per group. Analysis of variance F (2,18) ═ 4.971, P ═ 0.0191. Bottom: the bar graph compares the pure CK, unstimulated and GENUS stimulated CK-p25 mice. N-5 mice per group. Analysis of variance F (2,12) ═ 7.72, P ═ 0.0070.

In FIG. 10I, up-regulated genes in P301Stau and CK-P25 mice after chronic GENUS were selected based on the 60 gene clusters mediated by neurotransmitter transport and vesicles. Note that these genes were only up-regulated in CK-P25, P301S or both mice after GENUS (by metascape analysis) compared to non-stimulated mice. The protein-protein interaction network map (analyzed with Cytoscape V3.6.1 followed by Strings; enriched P value, 6.66E-15) is shown along with closely related other functionally enriched GO terms.

In FIG. 10J, S/T phosphoproteomic analysis was performed on visual cortex from unstimulated P301S tau-tg mice and 1 hour daily GENUS stimulation for 22 days. The heat map shows the differential phosphorylation proteins between non-stimulated and GENUS-stimulated P301S mice, which are involved in synaptic transmission and intracellular transport.

In fig. 10K, the heat map shows differentially phosphorylated proteins involved in synaptic transmission (chemical synaptic transmission, transsynaptic signaling) between non-stimulated and GENUS-stimulated CK-p25 mice. The name of the gene (corresponding protein) and the specific phosphate site are shown.

In fig. 10L, P301S tau mice were subjected to no stimulation or GENUS stimulation for 22 days. Total protein expression and S/T phosphorylated protein analysis were performed on visual cortex tissues using a tandem mass spectrometry tag (TMT)10-plex kit (see methods) and mass spectrometry (LC-MS/MS). N-3 wild-type inbred, 3 unstimulated P301S and 4 GENUS-stimulated P301S mice. Phosphorylated proteins with a fold change of ± 0.2 and an adjusted P-value <0.05 were considered statistically significant. GO terminology of biological processes associated with differential S/T phosphorylated proteins in CK-P25 and P301S tau mice compared to their respective control mice.

Fig. 10M-top: western blot shows pS774DNM-1, DNM-3 and GAPDH from naive CK, non-stimulated and 40Hz GENUS-stimulated CK-p25 mice. pS774DNM-1 levels in CK-p25 mice were significantly higher than in naive CK mice, while 40Hz GENUS significantly reduced pS774DNM-1 in CK-p25 mice. N-4 pure CK mice, 5 non-stimulated CK-P25 mice and 4 40Hz GENUS-stimulated CK-P25 mice (anova F (2,12) ═ 5.836, P ═ 0.021). Bottom: the bar graph shows the group differences. Note that this is a separate verification. Related to main plot 5F.

FIG 10N-top: western blot shows pS774DNM-1, total DNM-1, DNM-3 and GAPDH from wild type inbred, non-stimulated and 40Hz GENUS-stimulated P301S mice. Note that this is a separate verification. P < 0.05. Related to main plot 5F.

Fig. 11A-11J illustrate behavioral characterization of the impact of acute and chronic visual stimuli on a subject according to the disclosed inventive concepts.

Fig. 11A-top: a heat map showing occupancy of C57Bl6/J mice in the 10 minute compartment with or without GENUS is shown. There were no significant differences in exploratory behavior during the 10 minute baseline period prior to stimulation. N-6 mice per group. And T test, wherein T is 0.3173 and P is 0.7576. Similarly, no systematic change in velocity was detected throughout the 40Hz stimulation period when compared to the non-stimulated group. Bottom: the curves show the speed per minute over 30 minutes. Bidirectional repeated measurement analysis of variance: the search was performed over a period of 30 minutes, F (29,290) ═ 6.747, P < 0.0001; interaction between non-stimulated and GENUS-stimulated wild-type mice, F (29,290) ═ 0.9354, and P ═ 0.5652.

In FIG. 11B, C57Bl6/J mice (4 months OF age) were either not stimulated or stimulated with 40Hz light scintillation for 1 hour, then immediately injected and placed in OF. Left: the distance traveled after injection of tetrandrine indicated no difference between the GENUS group and the non-stimulated group. N-4 mice per group. Repeated measures analysis of variance F0.527 and P0.717. And (3) right: and (4) carrying out a Raxin score. Score 0, behavior is normal; score 1, immobility and stiffness; score 2, head swing; scoring 3, forelimb clonus and raising; scoring 4, continuously feeding and dropping; score 5, tonic clonic seizures; score 6, death. There was no significant difference in seizure susceptibility between the non-stimulated and GENUS groups. The analysis of variance F ═ 0.429 and P ═ 0.994 were repeated.

In fig. 11C, the NOR of wild type mice was tested 3 days after acclimation to the novel subjects. Left: each minute spent exploring familiar and novel subjects for a 30 minute duration. A novel object discrimination percentage is superimposed. The middle part: the histogram shows the cumulative (over 30 minutes) exploration of familiar and novel objects. Both non-stimulated and GENUS-stimulated mice spent significantly more time exploring novel subjects than familiar subjects. There were no differences between groups, indicating that acute 40Hz lamp flash exposure did not affect the ability of mice to discriminate novel subjects. N-6 mice per group. The cumulative preference of the novel object compared to the familiar object: no irritation, T (6) ═ 13.055, P ═ 0.00004; GENUS T (5) ═ 20.818, P ═ 0.000004. Novel object preferences between groups, T-test T-0.314 and P-0.760. And (3) right: histogram of total distance traveled, showing that GENUS does not alter the motor behavior of wild-type mice. The total distance moved, T-test T-0.5096 and P-0.6214.

In FIG. 11D, C57Bl6/J mice were subjected to no irritation or GENUS for 7 days, 1 hour per day. Mouse body weights were measured daily for 7 days 1 hour prior to the stimulation paradigm and 1 day after the stimulation regimen. N-14 non-stimulated mice and 12 GENUS-stimulated mice. The bar graph shows the body weight for 7 days. However, overall weight gain was achieved over all days in both groups (F (6,144) ═ 2.889, P ═ 0.011); there was no difference in body weight between the non-stimulated and GENUS-stimulated groups (F (6,144) ═ 1.327, P ═ 0.249).

However, figure 11E shows that there was a significant difference in body weight between wild type inbred and P301S tau mice (the inter-group two-way anova effect F (2,72) ═ 8.947, P ═ 0.0003); chronic GENUS (1 hour/day for 22 days) did not affect the body weight of P301S compared to non-stimulated P301S tau mice. N-13 wild-type inbred, 14 non-stimulated and 12 GENUS-stimulated P301S mice.

Fig. 11F shows that chronic GENUS did not affect body weight in CK-P25 mice compared to non-stimulated CK-P25 mice (inter-group two-way anova effect F (2,122) ═ 2.487, P ═ 0.0874). N-25 pure CK, 21 non-stimulated and 18 GENUS-stimulated CK-p25 mice.

Fig. 11G relates to the main pattern 6F. N is the same as in fig. 6F. The total distance traveled in the open field test between groups was significant. Analysis of variance F (2,36) ═ 10.27, P ═ 0.0003. However, there was no difference in GENUS-stimulated P301S mice compared to non-stimulated P301S (P > 0.99).

Fig. 11H relates to the main pattern 6B. N is the same as in fig. 6B. The total distance traveled in the open field test is shown. There was no difference in the total distance traveled during the open field test of non-stimulated and GENUS-stimulated CK-p25 mice. Analysis of variance F (2,49) ═ 0.01552, P ═ 0.9846.

FIG. 11I-left: there was no difference in plasma corticosterone levels between non-stimulated and 7-day GENUS-stimulated wild-type mice. Each group consisted of 12 mice. T is 1.17 and P is 0.255. The middle part: while inducing p25 in CK-p25 mice, mice were subjected to no stimulation or GENUS for 1 hour per day for 42 days. The number of mice per group is indicated in the graph. Plasma corticosterone levels were not different between naive CK, non-stimulated and GENUS-stimulated CK-P25 mice (F (2,35) ═ 0.4871, P ═ 0.6185). And (3) right: plasma corticosterone levels were also comparable in P301S tau mice (22 days stimulated) between wild type inbred, non-stimulated P301S and 22 days GENUS-stimulated P301S mice (F (2,35) ═ 0.6874, P ═ 0.5096). The number of mice in each group is given in the figure accordingly.

Fig. 11J relates to main pattern 7. In all experiments, mice were given the average speed of swimming in the Morris water maze test. Left: there were no significant differences between any of the groups in all 6 days of training of CK-p25 mice (fig. 7D). The interclass two-way anova effect F (2,252) ═ 0.3832, P ═ 0.6821. The middle part: there were no differences between groups in all 5 days of training of P301S tau mice (fig. 7H). The interclass two-way anova effect F (2,225) ═ 0.5726, P ═ 0.5651. And (3) right: there was a significant difference in the swimming speed of the 5XFAD mouse group (fig. 7I). Interclass two-way anova effect F (2,273) ═ 24.24, P < 0.0001. Multiple comparisons were made with the corrections of Bonferroni. There was no difference from day 1 to day 4. Day 5: wild type pure breed vs.5XFAD + has no stimulation, and P is more than 0.9999. Wild type pure breed vs.5XFAD +40Hz GENUS, P is 0.0022. 5XFAD + without stimulation vs.5XFAD +40Hz GENUS, P is 0.0483. Day 6: wild pure breed vs.5XFAD + has no stimulation, and P is 0.0005. Wild type pure breed vs.5XFAD +40Hz GENUS, P ═ 0.0037. 5XFAD + has no stimulation vs.5XFAD +40Hz GENUS, and P is more than 0.9999. Day 7: wild pure breed vs.5XFAD + has no stimulation, and P is less than 0.0001. Wild type pure breed vs.5XFAD +40Hz GENUS, P ═ 0.0043. 5XFAD + without stimulation vs.5XFAD +40Hz GENUS, P is 0.5716.

Fig. 12A-12C show that chronic visual stimulation at 80Hz does not affect the Morris water maze of 5XFAD mice according to the disclosed inventive concepts.

Figure 12A shows MWM performance of 5XFAD mice exposed to 80Hz stimulation for 22 days for 1 hour per day. The number of mice per group is indicated in the broken line legend. However, the latency of looking for the platform during training was not different between the non-stimulated and 80Hz stimulated 5XFAD groups (two-way anova F (2,180) ═ 13.33, P < 0.0001); both groups required more time to find the platform than the wild type inbred mice.

Fig. 12B indicates the number of times the platform crossed during probe testing. F (2,30) ═ 3.622, P ═ 0.0390. Multiple comparisons were made with the corrections of Bonferroni. P < 0.05.

Figure 12C shows that non-stimulated and 80Hz stimulated 5XFAD mice spent significantly less time in the target quadrant during the probe test compared to wild type inbred mice. F (2,30) ═ 5.643, P ═ 0.0083. Compared to wild-type, inbred mice, stimulated P-0.014, 80 Hz-P-0.0371, with Bonferroni corrected multiple comparisons. P < 0.05.

Details of the experiment and analysis

Start method

Animal model

All experiments were approved by the Massachusetts institute of technology, comparative institutional animal Care Committee. C57BL6, Tg (Camk2a-tTA), Tg (APPSwFlLon, PSEN 1M 146L L286V), Tg (Prnp-MAPT P301S) PS19 and Fos-tm1.1(cre/ERT2) were obtained from Jackson laboratories. Tg (tetO-CDK5R1/GFP) was generated in our laboratory. All transgenic mice were bred and housed in our animal facility.

The C57BL/6J mice used were 3, 10 or 17 months old. CK-control and CK-p25 (Camk2a-tTA hybridized with tetO-CDK5R1/GFP) mice were fed with doxycycline-containing chow. Normal rodent diets were given to induce p25-GFP transgene expression. Typically, p25 expression was induced for 6 weeks. At the beginning of the experiment, P301S tau mice were 7 months of age, while 5XFAD mice were 9 or 11 months. All mice were housed in groups (2-5 mice per cage) except for the mice implanted with the microdrive. All experiments were performed using age-matched litters. Two immunostaining and electrophysiology experiments were performed using mice to reach the total number defined in the legend. All behavioral experiments described herein (except the Morris water maze of young and old wild-type mice and tau P301S mice) were performed once using the total number of animals defined in the legend. Rather than using statistical methods to predetermine the sample size, we chose to use the group size from a similar previously released study in our laboratory setting (Iaccarino et al, 2016; not et al, 2016) to ensure that inter-group differences remain minimized. The block diagram in fig. 1 shows the median, the upper (75%) and lower (25%) quartile range. All plots with error bars in fig. 2 to 6 are expressed as mean ± SEM and all samples are expressed as number of mice (N), unless otherwise indicated.

40Hz lamp flicker stimulation

As before, light blinking stimulation was delivered (Iaccarino et al, 2016). The mice were transported from the storage room to a scintillation processing chamber located on the adjacent floor of the same building. Before the start of the experiment, mice were introduced into a test cage (similar to a household cage except without bedding and covered on three sides with black flakes) after 1 hour of acclimation in dim light. The mice were allowed to move freely within the cages, but had no food or water during the 1 hour of lamp blinking. An array of Light Emitting Diodes (LEDs) was present on the open side of the cage and was driven to flash at a frequency of 40Hz using the Arduino system in a square wave current mode. After 1 hour of 40Hz lamp flash exposure, mice were returned to their home cages and allowed to rest for an additional 30 minutes before being transferred back to the storage room. However, normal indoor light control mice were exposed to similar cages with similar food and water restrictions; they only experience normal room light.

Tissue preparation

Immunohistochemistry mice were endocardial perfused with 40ml ice-cold Phosphate Buffered Saline (PBS) and 4% paraformaldehyde in 40ml PBS. Brains were removed and fixed in 4% PFA overnight at 4 ℃ and then transferred to PBS before sectioning.

Western blotting: visual cortex was dissected and snap frozen in liquid nitrogen and stored in a freezer at-80 ℃ until processing. The samples were homogenized using a glass homogenizer with RIPA (50mM Tris HCl pH 8.0, 150mM NaCl, 1% Np-40, 0.5% sodium deoxycholate, 0.1% SDS) buffer. The concentration of protein in the sample was quantified using the Bio-Rad protein assay. An equal concentration of protein was prepared and SDS-sample buffer was added.

Immunohistochemistry

The brain was mounted on a vibrating microtome table (Leica VT1000S) using super glue and 40 μm sections were prepared. Sections were subsequently washed with PBS and blocked with 5% normal donkey serum prepared in PBS containing 0.3% Triton-X100(PBST) for 2 hours at room temperature. The blocking buffer was aspirated and the sections were incubated with the appropriate primary antibody (prepared in fresh blocking buffer) on a shaker at 4 ℃ overnight. Sections were then washed three times (10 min each) with blocking buffer and then incubated with AlexaFluor 488, 555, 594, or 647-conjugated secondary antibody for 2 hours at room temperature. Then three washes with blocking buffer (15 min each) and a final wash with PBS (10 min) and sections were fixed with fluromount-G (electron microscopy).

A combination of the following secondary antibodies was used: (1) alexa fluors 488, 594, and 647, (2) Alexa fluors 555 and 647, and (3) Alexa fluors 594 and 647.

Imaging and quantification

Images were acquired at the same settings under all conditions using LSM710 or LSM 880 confocal microscopes (Zeiss) with 5-, 10-, 20-, 40-or 63-fold objective lenses. Images were quantified using ImageJ 1.42q or imarisx 648.1.2 (Bitplane, zurich, switzerland). For each experimental condition, two coronal sections of a specified number of animals were used. The average of the two images per mouse was further used for quantification. Quantification is carried out by experimenters, as far as possible, who do not know the genotype and the treatment conditions.

NeuN, c-Fos and GFP positive cell counts: all images were taken and quantified in a Z-stack-10 per image. The average of every two and the sum of all counts were calculated using ImageJ. NeuN counts of P301S tau-tg and 5XFAD were performed by an experimenter blinded to the treatment conditions.

γ H2Ax positive cell count: the multi-point tool in ImageJ was used to count the cells.

vgut 1, basnoon point: an LSM 880 with a 63 objective lens and 3 zoom is used to acquire the image. The deep layers of the visual and somatosensory cortex (mainly 4 and 5), the CA1 layer of radiation, and the 5 th layer of the ventral cingulate cortex are all targeted. A single planar image is acquired. The particle count plug in ImageJ was used to quantify the number of vgout 1 points.

pS202/T205tau intensity: the LSM710 with 40 x objective lens was used to acquire the full slice thickness of the z-stack of 40 μm (40 images per field). All images were compressed/folded and the signal intensity was measured in ImageJ.

pS774-DNM1 Strength: the LSM 880 with 63 objective and 3 zoom was used to acquire the full slice thickness of the z-stack of 40 μm (40 images per field). All images were compressed/folded and the signal intensity of the spots was measured in ImageJ.

Plaque: the D54D2 antibody stained total plaque intensity and the number of plaques was quantified by an experimenter without knowledge of genotype and treatment conditions. The entire 40 μm section was z-stack imaged at 0.5 μm intervals, then they were combined and signal intensity was measured using ImageJ. Particle analysis tool in ImageJ was used at 5 μm²The threshold of (c) counts the number of plaques. All plaque intensity and plaque count counts were performed by experimenters who had no knowledge of the treatment conditions.

C1q strength: the LSM710 with 40 x objective lens was used to acquire the full slice thickness of the z-stack of 40 μm (40 images per field). All images were compressed/folded and the signal intensity was measured in ImageJ.

CD 40: the LSM 880 with 63 x objective lens was used to acquire the full slice thickness of the z-stack of 40 μm (40 images per field). All images were compressed/folded and the signal intensity was measured in ImageJ.

Lateral ventricle: the complete coronal section was imaged using an LSM710 microscope with a 5-fold objective at-2.0 frontal halogen spot (cranial to caudal). The free-hand selection tool of ImageJ was used to draw a contour covering the entire area of the lateral ventricle and measure the area of the LV.

Microglia: iba1 immunoreactive cells are considered microglia. The LSM710 with 40 x objective lens was used to acquire the full slice thickness of the z-stack of 40 μm (40 images per field). Imaris is used for 3D rendering of images to quantify the total volume of somatic cells and process microglia. The Iba1 aggregate analysis was performed in the Image J3D rendering plug-in. The minimum distance between Iba1 was calculated for each microglia from the images. All images were compressed/folded and the total number of Iba1 positive cells was quantified using Image J.

Thickness of the skin layer: the complete cortical column was imaged using an LSM710 microscope with a 5-fold objective. ImageJ was used to measure the distance between the outer cortical boundary and the cortical side of the corpus callosum.

Brain weight measurement: mice were sequentially perfused endocardially with PBS and 4% PFA, and then brains were fixed in 4% PFA overnight. The brains were washed with PBS to remove excess, and then weighed with a wet laboratory high precision scale (Mettler Toledo, accurate to 1 mg). Another group of mice was sacrificed and brains were snap frozen in liquid nitrogen and brain weight was measured. In each of these two independent indices, brain weight was normalized to pure CK brain.

Western blot

Six to eight micrograms of protein was loaded onto 6%, 8%, 10% or 15% polyacrylamide gels and electrophoresed. Proteins were transferred from acrylamide gels to nitrocellulose membranes (Bio-Rad) at 100V for 120 min. The membranes were blocked with milk (5% w/v) diluted in PBS containing 0.1% Tween-20(PBSTw) and then incubated overnight at 4 ℃ in primary antibody. The next day, they were washed three times with PBSTw and incubated with horseradish peroxidase-linked secondary antibody (GEHealthcare) for 60 minutes at room temperature. After three washes with PBSTw, the membrane was treated with a chemiluminescent substrate and developed. Signal intensity was quantified using ImageJ 1.46q and normalized to the value of a control protein (such as β -actin, GAPDH, or total protein assayed by the corresponding phosphorylated protein).

Micro-driver implantation and in vivo electrophysiology

Tungsten filament electrode driver: the microactuator was custom-built using a 3D print actuator base with a tungsten wire electrode (bare wire diameter 50 μ M, coating diameter 101.6; A-M system) and a Neuralynx electrode interface board (EIB-36) with a perfluoroalkoxy coating. The polyimide tube serves to protect the electrodes and reduce electrical noise. Electrodes were arranged to target layer 3 or layer 4 of the visual cortex (coordinates relative to the pro-halogen point, anterior-posterior (AP), -3.0; endo-exo (ML), +2.0), SS1(AP, -2.0; ML, +2.3), CA1 region of hippocampus (AP, -1.8; ML, +1.5) and cingulate gyrus region of prefrontal cortex (AP, + 1.0; ML, + 0.2). A reference electrode was placed in the cerebellum.

Quadrupole driver: the custom microactuator comprised four nichrome quadrupoles (14 mm; california thin line), gold plated (neuralnx), with a resistance of 200 to 250kU, implanted in a row (running along the CA3 to CA1 axes of the dorsal hippocampus) in the dorsal CA1(AP, -1.8). A reference electrode was placed in the fiber bundle above the hippocampus.

And (3) operation: mice were anesthetized with Abametin, restrained in a stereotaxic apparatus, and craniotomy was performed to expose the visual cortex, somatosensory cortex, and prefrontal cortex. The microdriver is implanted and slowly lowered to the target depth. Mice were allowed to recover for 4 days.

In vivo electrophysiology: after a 2-3 day dwell period, recordings were initiated, allowing the animals to move freely in a small open field. The recording phase consisted of 10-15 minutes, where the LED flashed at a frequency of 40Hz, but was completely enclosed by a black acrylic polypropylene sheet, which was then immediately removed for another 10 minutes, the sheet removed, and the animal exposed to the flashing LED. Data were acquired using a Neuralynx SX system (Neuralynx, Bozeman, MT, usa) and signals were sampled at 32,556 Hz. The position of the animal is tracked using a red light emitting diode fixed to the microactuator. At the end of the experiment, mice were terminally anesthetized and their anatomic location was confirmed by marking the electrode locations by electrolytic injury of brain tissue with 50 μ Α current separately for 10s through each electrode.

Peak value: the individual cells were isolated manually by drawing cluster boundaries around the 3D projection of the recorded spike, which is presented in the spikeport 3D software (Neuralynx). If the average spike width exceeds 200 μ s and the Complex Spike Index (CSI) is ≧ 5, the cell is considered a pyramidal neuron.

And (3) data analysis: the LFP is first filtered to the nyquist frequency of the target sampling rate and then downsampled 20 times (to 1628 Hz). Power spectral analysis was performed using the pwelch function in Matlab using a 500ms time window (50% overlap) and included only LFP data fragments with animal velocities maintained >4 cm/s. As previously described (Vinck et al, 2011), a weighted phase lag exponential (WPLI) (more appropriate for the coherence measure of small rodent brains) was used to eliminate any potential contamination of coherence by volume conduction signals. WPLI was calculated for paired electrodes in anatomically different regions only during periods of animal speed >4 cm/s. All analyses were performed using Matlab.

Pyramidal cell 40Hz modulation: the relationship between the peak firing time and the 40Hz LFP phase was calculated using a circular statistics toolbox as described previously (middlleton and McHugh, 2016). Briefly, the spikes are sorted and the LFP traces are filtered using a continuous wavelet transform (cmor 1.5-1 wavelet centered at 40 Hz), returning the phase and amplitude of the transient signal. The spike time is linearly interpolated to determine the phase, where the peak and valley of γ are defined as 0 degrees and 180 degrees, respectively. For each 20 degree interval, the resulting phase values are combined to generate a transmission probability. Cells were considered phase locked only if their distribution was significantly different from the uniform distribution (P <0.05 round rayleigh test), and the phase lock intensity was calculated as the average resulting length.

RNA sequencing

Isolation of microglia: visual cortex was quickly dissected and placed in ice-cold Hanks Balanced Salt Solution (HBSS) (Gibco, life technologies, cat. No. 14175-. The tissue was enzymatically digested using the neural tissue dissociation kit (P) (Miltenyi Biotec, catalog number 130-. Specifically, the tissue was enzymatically digested at 37 ℃ for 15 minutes instead of 35 minutes, and the resulting Cell suspension was passed through a 40 μm Cell filters (Falcon Cell filters, Sterile, Corning, product 352340) instead of a 70 μm MACS smartfilter. The resulting cell suspension was then stained using Allophycocyanin (APC) -conjugated CD11b mouse clone M1/70.15.11.5(Miltenyi Biotec, 130-098-088) and Phycoerythrin (PE) -conjugated CD45 antibody (BD Pharmingen, 553081) according to the manufacturer's recommendations (Miltenyi Biotec). FACS was then used to purify CD11b and CD45 positive microglia. Criteria, strict side scatter width and area, and forward scatter width and area were used to distinguish doublets from gated singlets only. Live cells were identified by staining with Propidium Iodide (PI) and gating only PI negative cells. CD11b and CD45 double positive cells were sorted into 1.5ml centrifuge tubes containing 500. mu.l of RNA lysis buffer (QIAGEN, Cat. 74134) containing 1% beta-mercaptoethanol (Sigma-Aldrich, Cat. No. M6250). RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN, Cat. No. 74134) according to the manufacturer's protocol. RNA was eluted and then stored at-80 ℃ until the entire transcriptome was amplified, library construction and sequencing.

Separation of neurons: the visual cortex was homogenized in 0.5mL ice-cold PBS with protease inhibitors and the suspension was centrifuged at 1600g for 10 min. The pellet was resuspended in 5mL NF-1 hypotonic buffer, incubated for 5 minutes, and then subjected to Dounce homogenization (pestle A) with 30 strokes. 5mL of NF-1 buffer was added to the suspension, and the pestle was washed with 10mL of NF-1 buffer, totaling 20 mL. All solutions were collected in 50 conical tubes and the homogenate was filtered through a 40 μm mesh filter. Nucleation at 3,000rpm (1,600x g) for 15 minutes. Resuspended in 30mL NF-1 buffer and mixed well. Shake down for 1 hour at 4 ℃. The pellet was washed once with 20mL NF-1 buffer, centrifuged at 3,000rpm for 15 minutes, and then resuspended in 2-5mL PBS + 1% BSA + protease inhibitor on ice for 20 minutes without disturbing the pellet. Alexa powder 488 or Alexa powder 647 conjugated NeuN antibody (1:500) was added to the tube and incubated at 4 ℃ for 20 minutes. Unbound antibody was washed from the suspension and centrifuged with PBS + 1% BSA + protease inhibitor. Nuclei were spun at 3,000rpm for 15 minutes, then resuspended in 0.5mL (PBS + protease inhibitor) and filtered with a 40 μm screen for FACS sorting. Two drops of nucblue liveready probe reagent (Thermo Fischer Scientific; Cat. No. R37605) were added for nuclear gating. NeuN positive cells were sorted into 1.5ml centrifuge tubes containing 500. mu.l of RNA lysis buffer (QIAGEN, Cat. No. 74134) containing 1% beta-mercaptoethanol (Sigma-Aldrich, Cat. No. M6250). RNA was extracted using the RNeasy PlusMini Kit (QIAGEN, Cat. 74134) according to the manufacturer's protocol. RNA was eluted and then stored at-80 ℃ until the entire transcriptome was amplified, library construction and sequencing.

Construction of RNA library: quality control of extracted total RNA was performed using an advanced analytical fragment analyzer prior to library construction. SMARTer chain Total RNA sequencing kit-Pico Input was used for the P301S neurons, CK-P25 neurons and P301S microglia RNA sequences. SMART sequencing v4 ultra-low input RNA kit was used for CK-p25 microglia-specific RNA sequencing. The library was constructed according to the manufacturer's instructions and sequenced on the Illumina Nextseq 500 platform at MIT biomicr center.

The original fastq data of 40bp paired-end sequencing reads were aligned to the mouse mm9 reference genome using STAR 2.4. The total number of reads and the percentage of reads aligned are as follows: CK-p25 neurons-total reading 18094674.857 ± 827050.464; the percentage of alignment was 85.323. + -. 0.414. P301S neurons-total reading 22792713.18 ± 6308817.636; the percentage of alignment was 84.45. + -. 0.548. CK-p25 microglia-total reading 28694964.5 ± 435841.6674; the percentage of alignment was 89.43 ± 0.25. [ P301S microglia-total reading 18990369.2. + -. 1667316.196; the percentage of alignment was 27.243 ± 4.04. This experiment needs to be further considered because the enantiomers are very low ]. Mapped reads were processed by cufflinks2.2(Trapnell et al, 2012) using mm9 reference gene annotation to estimate transcript abundance (for retention neuron data) for the library type fr-secondstrand. Gene differential expression testing between groups was performed using the Cuffdiff module with p-values <0.05 (for neuronal data). The geometric method was chosen as the library normalization method for Cuffdiff. For microglia data, gene exon counts were quantified from non-stranded RNA sequencing data using the featureCounts tool and statistical significance was calculated using DESeq 2. Color-coded scatter plots were used to visualize the FPKM set of values for differentially expressed genes and other genes.

Z-scores for repeat expression FPKM values of differentially expressed genes were seen in the heatmaps of the different sample groups. Gene ontology of microglia-specific DEG was performed using the Metascape tool, whereas gene ontology of neuron-specific DEG was performed using TOPPGENE

Proteomics and phosphoproteomics

Sample preparation, reduction, alkylation and trypsinization: the visual cortex was dissected and snap frozen in liquid nitrogen and stored in a freezer at-80 ℃ until further use. The samples were then homogenized with freshly prepared 8M urea solution using a plastic hand-held motor-driven homogenizer. The concentration of protein in the sample was quantified using the Bio-Rad protein assay. Samples containing 1mg protein per 1ml were prepared, aliquoted and stored in a freezer at-80 ℃ until further use. The protein was reduced with 10mM Dithiothreitol (DTT) for 1 hour at 56 deg.C, alkylated with 50mM iodoacetamide for 1 hour at Room Temperature (RT), and diluted with 100mM ammonium acetate at pH 8.9 to less than 1M urea. The proteins were digested overnight at room temperature using sequencing grade trypsin (Promega; 1. mu.g trypsin per 50. mu.g protein). The enzyme activity was quenched by acidifying the sample with acetic acid. The peptide mixture was desalted and concentrated on a C18Sep-Pak Plus column (Waters) and eluted with 50% acetonitrile, 0.1% formic acid and 0.1% acetic acid. The solvent was evaporated in a SpeedVac vacuum centrifuge. A 400 μ g aliquot of each sample was aliquoted and frozen in liquid nitrogen for 5 minutes, lyophilized and stored at-80 ℃.

TMT marking: TMT labeling and phosphopeptide enrichment: the lyophilized peptides were labeled with TMT-10-plex mass tag labeling kit (Thermo). For each TMT multiplex assay, a mixed sample consisting of equal amounts of peptides from wild-type, non-stimulatory and 40Hz entrained neurodegenerative mouse models was included, allowing relative quantification of the normalized channel. For TMT labeling, nine samples from 9 mouse peptide aliquots and one standard channel (400 μ g peptide per channel) were resuspended in 100 μ L70% (v/v) ethanol, 30% (v/v) 0.5M triethyl-ammonium bicarbonate pH 8.5, incubated with TMT reagent, and resuspended in 40 μ L anhydrous acetonitrile at room temperature for 1 hour. Samples were concentrated using a SpeedVac vacuum centrifuge, pooled and concentrated to dryness.

Peptide fractionation: the TMT-labeled peptide precipitate was separated by high pH reverse phase HPLC. Peptides were resuspended in 100uL buffer A (10mM TEAB, pH8) and separated on a 4.6mM x 250mM300 extended-C18, 5um chromatography column (Agilent) using buffer B (90% MeCN,10mM TEAB, pH8) at a flow rate of 1ml/min using a 90 minute gradient. The gradient is as follows: 1-5% B (0-10min), 5-35% B (10-70min), 35-70% B (70-80min), 70% B (80-90 min). Fractions were collected at intervals of 10 to 85 minutes (every 1 minute) over 75 minutes. These fractions were discontinuously divided into 15 fractions (1+16+31+46+61,2+17+32+47+62, etc.). The fractions were then subjected to a speed vacuum (Thermo Scientific Savant) to near dryness.

And (3) enrichment of phosphopeptides: phosphopeptides were enriched from each of the 15 fractions using the High-Select Fe-NTA phosphopeptide enrichment kit (Thermo) according to the manufacturer's instructions.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS): peptides were separated by reverse phase HPLC (Thermo EasynglC 1000) using a pre-column (home-made, 6cm 10 μm C18 column) and a self-assembled 5 μm tip analytical column (12cm 5 μm C18 column, new subject). Gradient elution was performed using a qxctive Plus mass spectrometer (Thermo) for 140 minutes before nanoelectrospray. Solvent a was 0.1% formic acid and solvent B was 80% MeCN/0.1% formic acid. The gradient conditions were 0-10% B (0-5 min), 10-30% B (5-105 min), 30-40% B (105-119 min), 40-60% B (119-124 min), 60-100% B (124-126 min), 100% B (126-136 min), 100-0% B (136-138 min), 0% B (138-140 min), and the mass spectrometer was operated in a data-dependent mode. The parameters of the full scan MS are: the resolution was 70,000 in the 350-2000m/z range, the AGC 3e6 and maximum IT was 50 ms. After a complete MS scan, MS/MS was performed, with the NCE of the first 10 precursor ions in each cycle being 34 and the dynamic exclusion time being 30 s. The original mass spectral data file (. raw) was searched using proteome findings (Thermo) and Mascot version 2.4.1(Matrix Science). Mascot search parameters are: the mass tolerance of the precursor ion is 10 ppm; the mass tolerance of fragment ions is 15 mmu; 2 leaky cleavage with trypsin; the immobilized modifications were carbamoylmethylation of cysteine and TMT 10plex modification of lysine and peptide N-terminus; the variable modifications are methionine oxidation, tyrosine phosphorylation and serine/threonine phosphorylation. TMT quantification was performed using proteomic findings and isotopic correction was performed according to the manufacturer's instructions, which were then normalized to the mean value for each TMT channel. Only peptides with Mascot scores greater than or equal to 25 and isolated interference less than or equal to 30 were included in the data analysis.

The washed pre-column was connected in series with an internally packed analytical capillary column [50 μm ID × 12cm packed with 5 μm C18 beads (YMC gel, ODSAQ, 12nm, S-5 μm, AQ12S05) ] with an integrated electrospray tip (-1 μm orifice). The polypeptide was eluted using a 140 minute (phosphopeptide) or 90 minute (total peptide) gradient of 9% to 70% acetonitrile in 0.2M acetic acid at a flow rate of 0.2ml/min with a split flow rate of-10,000: 1, resulting in a final electrospray flow rate of-20 nL/min. A total of 15 fractions were collected from each sample. The phosphopeptides were analyzed using a Thermo Q active Hybrid quadrupolode-Orbitrap Plus mass spectrometer set up as follows: spray voltage, 2 kV; no sheath or auxiliary air flow, and the temperature of the heated capillary tube is 250 ℃; the S-mirror radio frequency level is 50%. Q active operates in a data dependent acquisition mode. Full scan MS spectrum [ mass to charge ratio (m/z), 350 to 2000; based on the predicted AGC of the previous scan, after accumulating ions at the 3e6 target value, the resolution was detected in the Orbitrap analyzer, 70,000 at m/z 200. For each complete scan, the 15 strongest ions were separated by high energy collision dissociation (HCD) (separation width 0.4m/z) and fragmented (collision energy (CE): 32%), maximum injection time 300ms and resolution 35,000. Total peptide analysis was performed on an LTQ Orbitrap XL mass spectrometer with the following settings: spray voltage, 2 kV; without a sheath or auxiliary gas flow, the heated capillary temperature was 250 ℃. The analysis is performed in a data-dependent acquisition mode; the full scan mass spectrum (m/z range 400-. For each full scan, the 10 strongest ions were isolated in HCD cells (separation width 3Da) and fragmented by HCD (CE: 75%) and then detected in Orbitrap (ion target value 1x105) for iTRAQ marker ion quantification.

Mass spectrometry peptide mapping data analysis: the original mass spectral data file was loaded into proteome finding version 1.4.1.14(DBversion:79) (Thermo) and searched in the mouse SwissProt database using Mascot version 2.4(Matrix Science). TMT reporting quantification was extracted and isotopes were corrected in proteomic findings. The initial mass tolerance of the tandem mass spectrum to the precursor mass was 10ppm and the initial mass tolerance of the fragment ion was 15mmu matched. Cysteine carbamoylmethylation, TMT-tagged lysine and the N-terminus of the protein were searched for as fixed modifications. Phosphorylation of oxidized methionine, serine, threonine and tyrosine was searched as variable modifications. The minimum length of the peptide is seven amino acids. All peptide datasets were filtered by an ion fraction >20 to ensure high confidence in peptide identification and phosphorylation localization and to achieve peptide (FDR) below 1%. Based on the median relative peptide quantification obtained from the crude peptide analysis, phosphopeptide quantification was normalized to correct for subtle variations in sample size between TMT channels. For each phosphopeptide, relative quantification was expressed as the ratio between the TMT ion intensity from each analyzed sample and the included normalized channel.

Bioinformatics analysis: to identify the differentially expressed and phosphorylated peptide fragments that were significantly modulated in proportion, we selected an arbitrary cut-off value of ± 20% difference, adjusted P value < 0.05. The unregulated background library consisted of peptides in a ratio of 0.8-1.2. Thus, subsequent bioinformatic analysis included peptides with normalized channel ratios <0.8 and >1.2, respectively, relative to what was considered down-and up-regulated, respectively. Protein names from protein accession numbers were converted to gene lists using the Uniprot Id mapping search tool. The protein network was obtained by using the STRING database (version 10.5). Including all active interaction sources except text mining, and to ensure high confidence, confidence scores in excess of 0.9 are required. Gene Ontology (GO) term enrichment analysis was first performed on terms related to biological processes using STRING (http:// STRING-db. org), TOPPGENE (topgene. cchmc. org), and Metascape (http:// Metascape. org) bioinformatics resources, followed by manual filtration to find common terms from these three resources. GO terms obtained in TOPPGENE are reported. For each individual mouse strain (C57BL6/J, CK-P25, P301S tau-tg, and 5XFAD), a gene set was analyzed that included derivation from each pool of differentially regulated total peptides or S/T phosphopeptides (up and down regulation).

Behavior

In an open field: mice were introduced into an open field box (size: 460mm long, 460mm wide, 400mm high; TSE system) and tracked for 5 minutes using noldus (ethovision), measuring the time spent in the center and peripheral regions of the field.

Elevated Plus Maze (EPM): mice were introduced into the central region of EPM (any maze size: arm length 35cm, width 5cm) and followed for 10 minutes using the Noldus program. The time spent by each branch and EPM center is calculated off-line.

Novel object recognition of CK-P25 and P301S tau mice: mice were introduced into the open field and the time spent in the center of the field was calculated. The next day, the mice were reintroduced into the same open field box, which now contains two more familiar subjects (but the novel object will be familiar at the next stage), and allowed to explore the subjects for 10 minutes. Thereafter, they returned to the same venue 10 minutes after the last exploration, with one of the two objects replaced by the new object. The behavior of the mice was monitored for 7 minutes. The time spent exploring familiar and novel objects was recorded using Noldus and calculated off-line.

Susceptibility to seizures: mice were injected with tetrandrine (i.p.) and placed in open field and recorded for 30 minutes using a Noldus and side-mounted camera. The distance traveled was calculated offline in Noldus and seizure severity was scored manually.

Morris water maze: MWM is a test used to evaluate spatial learning. The MWM relies on visual cues to navigate from a starting position around the open swimming stadium to find an underwater escape platform. The apparatus consisted of a circular cell (122 cm diameter) filled with tap water (22-24 ℃) and added non-toxic white paint to make the solution opaque. The escape platform (10 cm diameter) was provided with blunt protruding edges for better gripping when submerged 1 inch below the water level. The pool is divided into four equal quadrants, labeled N (North), E (east), S (south), and W (West), respectively. Throughout the experiment, mice were introduced into the maze in random order starting from the edge and the experiment was performed for 60 seconds. The time required to find the hidden platform (latency) is recorded. The probe test was performed 24 hours after the last training trial and the immersion platform was removed. All training and probe testing trials were recorded using Noldus.

CK-P25, P301S, and 5XFAD mice were subjected to GENUS in the morning (up to 12 pm) and tested in MWM in the afternoon (3 pm to 7 pm), which limited the behavioral testing time to only 4 hours per day. We ensure that at least two and at most 4 trials per day are used during training, allowing us to be able to run all groups in MWM while still maintaining strict dark/light cycles. The total number of training steps for all AD mice used for MWM was comparable, although the number of training days varied.

Statistical analysis

Statistical analysis was performed in SPSS, Matlab or Prism. The sample size was not predetermined. Statistical significance was calculated using independent sample t-test, Wilcoxon Rank-sum test, one-way anova or two-way repeated measures anova, and Bonferroni post hoc analysis and Krushkal Wallis test. Statistical significance was set to 0.05.

Conclusion

Inventive aspects of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods (if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent) is included within the inventive scope of the present disclosure.

Moreover, various inventive concepts may be embodied as one or more methods, examples of which have been provided. The actions performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed which perform acts in an order different than illustrated, which may include performing some acts concurrently, even though shown as sequential acts in the illustrative embodiments.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

All definitions, as defined and used herein, should be understood to control dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

As used herein, the terms "about" and "approximately," when used in conjunction with a numerical value and/or range, generally refer to those numerical values and/or ranges that are close to the recited numerical values and/or ranges. In some instances, the terms "about" and "approximately" can mean within ± 10% of the stated value (including the value itself). For example, in some cases, "about 100[ units ]" may mean within ± 10% of 100 (e.g., from 90 to 110). In other instances, the terms "about" and "approximately" can mean within ± 5% of the stated value (including the value itself). In other instances, the terms "about" and "approximately" can mean within ± 1% of the stated value (including the value itself). The terms "about" and "approximately" may be used interchangeably.

The indefinite articles "a" and "an" as used herein in the specification and in the claims are to be understood as meaning "at least one" unless explicitly indicated to the contrary.

The phrase "and/or" as used in the specification and claims should be understood to mean "either or both" of the elements so combined, i.e., elements that are present together in some cases and not continuously present in other cases. Multiple elements listed with "and/or" should be interpreted in the same manner, i.e., "one or more" of the elements so combined. In addition to the elements specifically identified by the "and/or" clause, other elements may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, when used in conjunction with open-ended language (e.g., "including"), reference to "a and/or B" may refer in one embodiment to a alone (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than a); in yet another embodiment, both a and B (optionally including other elements); and so on.

As used herein in the specification and claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when items in a list are separated, "or" and/or "should be interpreted as being inclusive, i.e., including at least one (and also more than one) of the plurality or list of elements, and optionally other unlisted items. Merely explicitly stating the opposite terms, such as "only one" or "exactly one", or "consisting of …" when used in the claims will refer to comprising exactly one element of a plurality or list of elements, for example. In general, the term "or" as used herein should only be construed to indicate an exclusive alternative (i.e., "one or the other, but not both") before exclusive terms such as "either," one, "" only one, "or" exactly one. "consisting essentially of …" when used in the claims shall have its ordinary meaning as used in the patent law field.

As used herein in the specification and claims, the phrase "at least one," when referring to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that, in addition to the elements specifically identified in the list of elements to which the phrase "at least one" refers, other elements are optionally present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, in one embodiment, "at least one of a and B" (or, equivalently, "at least one of a or B" or, equivalently "at least one of a and/or B") can refer to at least one, optionally including more than one, a, with no B present (and optionally including elements other than B); in another embodiment, may refer to at least one, optionally including more than one, B, with no a present (and optionally including elements other than a); in yet another embodiment, may refer to at least one, optionally containing more than one a, and at least one, optionally containing more than one B (and optionally containing other elements); and so on.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," including, "" carrying, "" having, "" containing, "" involving, "" containing, "" consisting of … and the like are to be understood to be open-ended, i.e., to mean including but not limited to. As described in the united states patent office patent examination program manual, section 2111.03, only the transition phrases "consisting of …" and "consisting essentially of …" should be closed or semi-closed transition phrases, respectively.

Claims (86)

1. A method for treating dementia or alzheimer's disease in a subject in need thereof, the method comprising the steps of:

A) non-invasively delivering to the subject a chronic visual stimulus having a frequency of about 30Hz to about 50Hz to entrain synchronized gamma oscillations in multiple brain regions of the subject, the multiple brain regions including at least the prefrontal cortex (PFC) and hippocampus of the subject.

2. The method of claim 1, wherein in a), the chronic visual stimulus has a frequency of about 35Hz to about 45 Hz.

3. The method of claim 2, wherein in a), the chronic visual stimulus has a frequency of about 40 Hz.

4. The method of claim 3, wherein A) comprises causing a Local Field Potential (LFP) at about 40Hz in at least the prefrontal cortex and hippocampus of the subject.

5. The method of claim 2, wherein in a), the chronic visual stimulus has a duty cycle of 50%.

6. The method of claim 2, wherein a) comprises driving an array of Light Emitting Diodes (LEDs) with a square wave current signal to produce the chronic visual stimulus.

7. The method of claim 6, wherein:

the square wave current signal has a duty cycle of 50%; and is

The frequency of the square wave current signal is equal to or about 40 Hz.

8. The method of claim 7, wherein A) comprises causing a Local Field Potential (LFP) at about 40Hz in at least the prefrontal cortex and hippocampus of the subject.

9. The method of claim 2, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for more than seven days.

10. The method of claim 9, wherein a) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 22 days.

11. The method of claim 10, wherein a) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 42 days.

12. The method of claim 10, wherein:

A) comprising driving an array of Light Emitting Diodes (LEDs) with a square wave current signal to produce the chronic visual stimulus;

the square wave current signal has a duty cycle of 50%; and is

The frequency of the square wave current signal is equal to or about 40 Hz.

13. The method of claim 12, wherein a) comprises causing a Local Field Potential (LFP) at about 40Hz in at least the prefrontal cortex and hippocampus of the subject.

14. The method of claim 13, wherein the plurality of brain regions of the subject comprises a visual cortex, a somatosensory cortex, a hippocampus, and a prefrontal cortex of the subject.

15. The method of claim 1, wherein a) comprises:

A1) non-invasively delivering the chronic visual stimulus having a frequency of about 30Hz to about 50Hz to the subject to simultaneously entrain synchronized gamma oscillations in the plurality of brain regions of the subject, the plurality of brain regions including at least the prefrontal cortex and hippocampus of the subject.

16. The method of claim 15, wherein a1) comprises:

significantly increasing gamma coherence having a frequency between 30Hz to 50Hz between the plurality of brain regions of the subject, the plurality of brain regions including at least the prefrontal cortex and hippocampus of the subject.

17. The method of claim 16, wherein the plurality of brain regions of the subject comprises a visual cortex, a somatosensory cortex, a hippocampus, and a prefrontal cortex of the subject.

18. The method of claim 17, wherein in a), the chronic visual stimulus has a frequency of about 35Hz to about 45 Hz.

19. The method of claim 18, wherein in a), the chronic visual stimulus has a frequency of about 40 Hz.

20. The method of claim 16, wherein a) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for more than seven days.

21. The method of claim 20, wherein a) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 22 days.

22. The method of claim 21, wherein a) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 42 days.

23. The method of claim 22, wherein:

A) comprising driving an array of Light Emitting Diodes (LEDs) with a square wave current signal to produce the chronic visual stimulus;

the square wave current signal has a duty cycle of 50%; and is

The frequency of the square wave current signal is equal to or about 40 Hz.

24. The method of claim 15, wherein a1) further comprises:

A2) non-invasively delivering the chronic visual stimulus with a frequency of about 30Hz to about 50Hz to modulate neuronal activity between the plurality of brain regions of the subject, the plurality of brain regions including at least the prefrontal cortex and hippocampus of the subject.

25. The method of claim 24, wherein a2) further comprises:

non-invasively delivering the chronic visual stimulus with a frequency of about 30Hz to about 50Hz to coordinate neuronal activity between the plurality of brain regions of the subject, the plurality of brain regions including at least the prefrontal cortex and hippocampus of the subject.

26. The method of claim 25, wherein the plurality of brain regions of the subject comprises a visual cortex, a somatosensory cortex, a hippocampus, and a prefrontal cortex of the subject.

27. The method of claim 26, wherein in a), the chronic visual stimulus has a frequency of about 35Hz to about 45 Hz.

28. The method of claim 27, wherein in a), the chronic visual stimulus has a frequency of about 40 Hz.

29. The method of claim 24, wherein a) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for more than seven days.

30. The method of claim 25, wherein a) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 22 days.

31. The method of claim 26, wherein a) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 42 days.

32. The method of claim 27, wherein:

A) comprising driving an array of Light Emitting Diodes (LEDs) with a square wave current signal to produce the chronic visual stimulus;

the square wave current signal has a duty cycle of 50%; and is

The frequency of the square wave current signal is equal to or about 40 Hz.

33. The method of claim 24, wherein a2) further comprises:

A3) non-invasively delivering the chronic visual stimulus with a frequency of about 30Hz to about 50Hz to reduce neurodegeneration in the plurality of brain regions of the subject, the plurality of brain regions comprising at least the prefrontal cortex and hippocampus of the subject.

34. The method of claim 33, wherein a3) further comprises:

non-invasively delivering the chronic visual stimulus with a frequency of about 30Hz to about 50Hz to reduce amyloid plaques in the plurality of brain regions of the subject, the plurality of brain regions including at least the prefrontal cortex and hippocampus of the subject.

35. The method of claim 33, wherein a3) further comprises:

non-invasively delivering the chronic visual stimulus with a frequency of about 30Hz to about 50Hz to reduce tau hyperphosphorylation in the plurality of brain regions of the subject, the plurality of brain regions including at least the prefrontal cortex and hippocampus of the subject.

36. The method of claim 33, wherein a3) further comprises:

non-invasively delivering the chronic visual stimulus with a frequency of about 30Hz to about 50Hz to reduce loss of neurons and synapses in the plurality of brain regions of the subject, the plurality of brain regions comprising at least the prefrontal cortex and hippocampus of the subject.

37. The method of claim 33, wherein a3) further comprises:

non-invasively delivering the chronic visual stimulus with a frequency of about 30Hz to about 50Hz to reduce brain atrophy in the plurality of brain regions of the subject, the plurality of brain regions including at least the prefrontal cortex and hippocampus of the subject.

38. The method of claim 33, wherein a3) further comprises:

non-invasively delivering the chronic visual stimulus with a frequency of about 30Hz to about 50Hz to reduce ventricular dilation in the plurality of brain regions of the subject, the plurality of brain regions including at least the prefrontal cortex and hippocampus of the subject.

39. The method of claim 33, wherein a) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for more than seven days.

40. The method of claim 39, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 22 days.

41. The method of claim 40, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 42 days.

42. The method of claim 41, wherein:

A) comprising driving an array of Light Emitting Diodes (LEDs) with a square wave current signal to produce the chronic visual stimulus;

the square wave current signal has a duty cycle of 50%; and is

The frequency of the square wave current signal is equal to or about 40 Hz.

43. The method of any one of claims 34-38, wherein a) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for more than seven days.

44. The method of claim 43, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 22 days.

45. The method of claim 44, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 42 days.

46. The method of claim 45, wherein:

A) comprising driving an array of Light Emitting Diodes (LEDs) with a square wave current signal to produce the chronic visual stimulus;

the square wave current signal has a duty cycle of 50%; and is

The frequency of the square wave current signal is equal to or about 40 Hz.

47. The method of claim 24, wherein a2) further comprises:

A3) non-invasively delivering the chronic visual stimulus with a frequency of about 30Hz to about 50Hz to reduce neuroinflammation of the plurality of brain regions of the subject, the plurality of brain regions including at least the prefrontal cortex and hippocampus of the subject.

48. The method of claim 47, wherein A3) further comprises:

A4) non-invasively delivering the chronic visual stimulus with a frequency of about 30Hz to about 50Hz to reduce an immune response of at least some microglia in the plurality of brain regions of the subject, the plurality of brain regions including at least the prefrontal cortex and hippocampus of the subject.

49. The method of claim 48, wherein A4) further comprises:

non-invasively delivering the chronic visual stimulus with a frequency of about 30Hz to about 50Hz to morphologically transform the at least some microglia in the plurality of brain regions of the subject, the plurality of brain regions including at least the prefrontal cortex and hippocampus of the subject.

50. The method of claim 48, wherein A4) further comprises:

non-invasively delivering the chronic visual stimulus with a frequency of about 30Hz to about 50Hz to increase protein degradation of the at least some microglia in the plurality of brain regions of the subject, the plurality of brain regions including at least the prefrontal cortex and hippocampus of the subject.

51. The method of claim 48, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for more than seven days.

52. The method of claim 51, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 22 days.

53. The method of claim 52, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 42 days.

54. The method of claim 53, wherein:

A) comprising driving an array of Light Emitting Diodes (LEDs) with a square wave current signal to produce the chronic visual stimulus;

the square wave current signal has a duty cycle of 50%; and is

The frequency of the square wave current signal is equal to or about 40 Hz.

55. The method of claim 24, wherein a2) further comprises:

non-invasively delivering the chronic visual stimulus with a frequency of about 30Hz to about 50Hz to improve genes and proteins involved in aberrant modification of at least one of membrane trafficking, intracellular transport, synaptic function, neuroinflammation, apoptotic processes, and DNA damage in the plurality of brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject.

56. The method of claim 15, wherein a1) further comprises:

A2) non-invasively delivering the chronic visual stimulus with a frequency of about 30Hz to about 50Hz to enhance learning and memory of the subject.

57. The method of claim 56, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for more than seven days.

58. The method of claim 57, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 22 days.

59. The method of claim 58, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 42 days.

60. The method of claim 59, wherein:

A) comprising driving an array of Light Emitting Diodes (LEDs) with a square wave current signal to produce the chronic visual stimulus;

the square wave current signal has a duty cycle of 50%; and is

The frequency of the square wave current signal is equal to or about 40 Hz.

61. A method for treating dementia or alzheimer's disease in a subject in need thereof, the method comprising the steps of:

A) non-invasively delivering to the subject a chronic visual stimulus having a frequency of about 30Hz to about 50Hz to entrain synchronized gamma oscillations in multiple brain regions of the subject, the multiple brain regions including at least the prefrontal cortex (PFC) and hippocampus of the subject, wherein A) comprises non-invasively delivering to the subject the chronic visual stimulus having a frequency of about 30Hz to about 50 Hz:

A1) simultaneously entraining synchronized gamma oscillations in the plurality of brain regions of the subject, the plurality of brain regions including at least the prefrontal cortex and hippocampus of the subject;

A2) coordinating neuronal activity between the plurality of brain regions of the subject, the plurality of brain regions including at least the prefrontal cortex and hippocampus of the subject;

A3) reducing neurodegeneration in the plurality of brain regions of the subject, the plurality of brain regions comprising at least the prefrontal cortex and hippocampus of the subject;

A4) reducing neuroinflammation in the plurality of brain regions of the subject, the plurality of brain regions including at least the prefrontal cortex and hippocampus of the subject;

A5) genes and proteins that ameliorate abnormal modifications involving at least one of membrane trafficking, intracellular transport, synaptic function, neuroinflammation, apoptotic processes, and DNA damage in the plurality of brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject; and

A5) enhancing learning and memory of the subject.

62. The method of claim 61, wherein A) comprises causing a Local Field Potential (LFP) at about 40Hz in at least the prefrontal cortex and hippocampus of the subject.

63. The method of claim 62, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 22 days.

64. The method of claim 63, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 42 days.

65. The method of claim 63, wherein:

A) comprising driving an array of Light Emitting Diodes (LEDs) with a square wave current signal to produce the chronic visual stimulus;

the square wave current signal has a duty cycle of 50%; and is

The frequency of the square wave current signal is equal to or about 40 Hz.

66. The method of claim 61, wherein:

A) comprising driving an array of Light Emitting Diodes (LEDs) with a square wave current signal to produce the chronic visual stimulus;

the square wave current signal has a duty cycle of 50%; and is

The frequency of the square wave current signal is equal to or about 40 Hz.

67. A method for treating dementia or alzheimer's disease in a subject in need thereof, the method comprising the steps of:

A) non-invasively delivering a chronic visual stimulus having a frequency of about 30Hz to about 50Hz to the subject to entrain synchronized gamma oscillations in multiple brain regions of the subject and improve aberrantly modified genes and proteins in degenerating neurons in multiple brain regions of the subject.

68. The method of claim 67, wherein the aberrantly modified genes and proteins in degenerating neurons in the plurality of brain regions of the subject are involved in at least one of membrane trafficking, intracellular transport, synaptic function, neuroinflammation, apoptotic processes, and DNA damage.

69. The method of claim 68, wherein the aberrantly modified genes and proteins comprise genes from at least the NueN positive neuronal nucleus in the visual cortex of the subject.

70. The method of claim 69, wherein A) comprises non-invasively delivering the chronic visual stimulus to reduce the loss or maintain the percentage of NueN-positive neuronal nuclei in at least the visual cortex of the subject.

71. The method of claim 68, wherein the aberrantly modified genes and proteins comprise S/T phosphorylated proteins, and wherein A) comprises non-invasively delivering the chronic visual stimulus to modify the S/T phosphorylated proteins to reduce S/T phosphorylation in at least the visual cortex of the subject.

72. The method of claim 71, wherein the S/T phosphorylated protein comprises dynamin1(DNM-1), and wherein A) comprises non-invasively delivering the chronic visual stimulus to reduce Ser774 phosphorylation in DNM-1.

73. The method of claim 68, wherein the aberrantly modified genes and proteins comprise vesicular glutamate transporter 1(vGlut1) having substantially reduced spots, and wherein A) comprises non-invasively delivering the chronic visual stimulus to significantly increase vGlut1 spots in the plurality of brain regions of the subject.

74. The method of claim 73, wherein the plurality of brain regions of the subject comprises a visual cortex, a somatosensory cortex, a hippocampus, and a prefrontal cortex of the subject.

75. The method of claim 67, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for more than seven days.

76. The method of claim 75, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 22 days.

77. The method of claim 76, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 42 days.

78. The method of claim 77, wherein:

A) comprising driving an array of Light Emitting Diodes (LEDs) with a square wave current signal to produce the chronic visual stimulus;

the square wave current signal has a duty cycle of 50%; and is

The frequency of the square wave current signal is equal to or about 40 Hz.

79. A method for treating dementia or alzheimer's disease in a subject in need thereof, the method comprising the steps of:

A) non-invasively delivering to the subject a chronic visual stimulus having a frequency of about 30Hz to about 50Hz to simultaneously entrain synchronized gamma oscillations in multiple brain regions of the subject to substantially increase gamma coherence between the multiple brain regions of the subject having a frequency between 30Hz to 50 Hz.

80. The method of claim 79, wherein the plurality of brain regions of the subject comprises a visual cortex, a somatosensory cortex, a hippocampus, and a prefrontal cortex of the subject.

81. The method of claim 80, wherein A) comprises causing a Local Field Potential (LFP) at about 40Hz in at least the prefrontal cortex and hippocampus of the subject.

82. The method of claim 79, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for more than seven days.

83. The method of claim 82, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 22 days.

84. The method of claim 83, wherein A) comprises non-invasively delivering the chronic visual stimulus for at least 1 hour per day for at least 42 days.

85. The method of claim 84, wherein:

A) comprising driving an array of Light Emitting Diodes (LEDs) with a square wave current signal to produce the chronic visual stimulus;

the square wave current signal has a duty cycle of 50%; and is

The frequency of the square wave current signal is equal to or about 40 Hz.

86. The method of claim 85, wherein a1) further comprises:

non-invasively delivering the chronic visual stimulus with a frequency of about 30Hz to about 50Hz to coordinate neuronal activity between the plurality of brain regions of the subject.

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