D409V/D409V KI, GBA enzyme assay, conditional / nGD models, animal models comparison, Saposin C dose

D409V/D409V KI

D409V/D409V KI (Jackson Laboratory Stock # 019106), harbors a point mutation at residue 409 in the murine Gba gene (this was a MJFF Tools Consortium). These mice express the mutant D427V Gba protein, which corresponds to the D409V mutation in the mature human GBA protein, and possess loxP sites flanking exons 6 through 8. When these mutant mice are bred to mice that express Cre recombinase, resulting offspring will have exons 6 through 8 deleted in the cre-expressing tissues and will no longer express the mutant D427V protein.

Cortex GlcSph plot row labels: D409V WT, D409V Homo, L444P WT, L444P Homo. Annotation <10%, (2015 MJF).

Paper)

i. GBA activity in Brain (20% of control, 2017 Sardi Venglustat, 6m old mice인듯)

ii. no GlcCer accumulation (=0||) (whole brain, 2017 Sardi venglustat)

iii. GlcSph (brain): 100x - 1000x (2015 mjf), greatly accumulation (cortex, 2017 Sardi venglustat)

iv. [A-syn]: i) ↑ in hippocampus but not in SN, but, D409V mutation is not reported in GD (D409H is a few, though) and this animal model does not typically manifest Gaucher features. ii) [2017 Sardi Venglustat]: ↑ proteinase K resistant a-syn의 aggregate (at least in hippocampus). (Total (mono+oligo)도 봤다는듯) iii) 2021 WORLDSymposium Matthews: at 12 m old, proteinase-K-resistant-aSyn in hippocampus

v. 2021 WORLDSymposium Matthews: at 12 m old, ↑ tau, (whole brain) inflammation (ie GFAP, IBA1)

GBA enzyme assay

In house) brain GBA activity is ~50% (Fuji & Axlead similar results)

RD-DDU:Gba D409V-loxP, Crosssite meeting 202012, p15, Brain GlcSph가 L444P보다 꽤 높음. 그런데, rhGCB 투이후 오히려 ↑(26.9%)

GBA enzyme assay caption:

Whole cortex of male mice at 3 mo Animals: GBA D409V KI wild and homo mice, 17 weeks, male Samples: Left cortex slices shown as 1 in the right figure (N=4/group) GBA enzyme assay: Follow the protocol provided by Fujii-san.

GCase activity bar chart figure caption: GCase activity (nmol/hr/mg protein), groups Wild, Homo, Results are Mean ± SEM (N=4). ***p<0.001 vs wild mice by Student's t-test. The GCase activity in the cortex of homo mice was significantly lower than that of wild mice.

MJFF: GBA D409VKi (hom) and crossed them with thy-1 aSyn or introduced PFF

For GBA animal models, the manuscript that we are putting together will likely be published only in mid-2021. However, in a nutshell, we used the GBA D409VKi (hom) and crossed them with thy-1 aSyn or introduced PFF and we noticed increased p129 syn pathology at 12 months in the GBAXthy1 syn but not in the PFF model. If you have additional questions regarding this data, I shall be happy to connect you with my colleague Nicole Polinski who led this work.

우리 GBA activator project에는 안 relevant!

nGD model / conditional

nGD model	(Jackson, 2019 #939) 좀더 aggressive 한 모델 근본이 D409V/409V 이니 우리 GBA activator project에는 안 relevant!
Method	intracerebroventricular administration of an AAV encoding a microRNA to Gba (AAV-GFP-miR-Gba) into D409V/D409V mouse. → significantly reduced GCase activity and increased substrate accumulation in the CNS, ↓ cognition, hyperactivity, ↓ gait, head retroflexion
conditional	Mx1-Cre-loxP system, Cre-mediated conditional deletion of GBA gene upon treatment with polyinosinic-polycytidylic acid, which activates the Mx1 promoter
conditional / result	a normal life span; vs however, there is no distinct CNS abnormality or glucosylceramide accumulation in the brain
conditional	Nestin-flox/flox (Calabresi et al., 2014), K14-Inl/Inl (Calabresi et al., 2014), and Kn-9H mice
conditional / result	These mice show neuronal loss, microglia activation, and glucosylceramide accumulation in the brain and develop neurological symptoms including seizures and paralysis However, the rapid and extremely severe disease development and death within 2-3 weeks
D409V/null	(FDA Eliglustat review) D409V/null mice are a heterozygous model of Type 1 Gaucher. Glucosylceramide in these mice begins to accumulate starting at three months of age and Gaucher cells begin to appear by four months of age. At year one, GL-1 in peripheral tissues is generally 16-fold above levels in wild type mice, and Gaucher cells are present in large numbers, particularly in the liver and lung. There is no accumulation of GL-1 in the brain, and the mice have a normal life span.
D409V/WT	(FDA Eliglustat review): normal levels of GL-1, do not form Gaucher cells, and have a normal life span
D409V/WT	(Clarke, 2019 #940) ↓ Cognition (at 12 m), no LBs (hippocampus), altered cholinergic machinery, ↑ gliosis,

Animal models comparison

Type of models	Model generation	Residual GBA activity in the brain	Substrate accumulation (brain)	Life span	Brain pathology	Remark
Enquist (KO mouse 임. K14-Inl/Inl Mice) (2007, Sweden)		Homo: 10% (fig 1E) Hetero: 80% of WT (fig 1E)	(Enquist: homo, hetero 모두 ↑ by (fig 1F) thousands fold	14 days	<Enquist> Neuronal loss in cortex & thalamus (Nissl) Astrogliosis (IBA-1 or MAC2) GFAP SI Figure 8 visualized but not quantified Massive microgliosis	D409V mutation is not reported in human GD
Sardi (2011, US)		Gba ?D409V/D409V: 25% KO:<10% Hetero:72%
Farfel-Becker (2014, Israel)		absolute number shown by areas but w/o control value	↑ different regions
Cabrera-Salazar (2010)		(24h post injection)
Osellame (2014) (= Enquist: KO mouse 임, ie (K14-Inl/Inl Mice))		↓ (Osellame) Homo: 27% Hetero: 43% of WT	(Fig 2) A-syn WB: midbrain section (Fig 2b IHC 에서 a-syn 이 heteroz 에선 축적 안 보였다고 했음)

a-syn Western blot (Osellame midbrain section)

fraction	WT	GBA KO heteroz	GBA KO homoz
TBS soluble		내가 봐서 별 차이없음.	차이있단언급없고 내가봐도 별 차이 없음.
(continuation)		다 안 보임
Insoluble (SDS/Urea soluble)		내가 봐서 별 차이없음.	차이있단언급없고 내가봐도 별 차이 없음.
(continuation)		내가 봐서 별 차이없음.	↑

4L/PS-NA mouse model (Mazzulli)

fraction	WT	GBA KO (?)
TritonX soluble
Insoluble		약간 ↑

Saposin C / V394L crossbreeding

• Crossbreeding of mice homozygous for the V394L mutation with the saposin C null mice
• Die <48h
• The accumulation of p62 and LAMP-2 (by IF)

Ginns 2014, US (L444P or R463C point mutation mouse)

• L444P or R463C point mutation mouse). Gba sequence가 TGACTTGGA에서 TGACCCGGA로 변경
• Residual activity: 35% - 65% of WT (2 days post injection); (tissue extracts ref [44-46]); Hetero는 안 쓰인 것 같음.
• Life span: >1 year
  1. TH+ cell not assessed
  2. Astrocytosis (quantified by GFAP WB)
  3. a-syn accumulation in striatum (IF, not Q)

Cullen (2011, Canada) D409Vgba knockin mouse model

• Normal life span
• 옆의 결과가 brain stem에서는 모두 차이없었음. (Residual GCase activity >20%,)
• Hetero의 gba activity는 불명

Hippocampus a-syn ELISA (Cullen)

	GBA Homozygous	GBA Heteroz
cytosolic	Only a trend for increase	No difference
TritonX soluble (memb)	↑ (25% increase, wt보다)	Not done
Insoluble	Only a trend for increase	Not done

결국, a-syn 은 12month homoz hippocampus에서만 올라가고, 12month homoz brain stem, 12month hetero, 12 week homo에선 안 올라갔음.

Stephens (1978)

(6-8 weeks old BALB/c male mice) nothing done to Saposin C

	7.5mg/kg single	10mg/kg single	10mg/kg daily, 4 wks	25mg/kg daily, 4 wks	50mg/kg daily, 4 wks	100mg/kg daily, 4 wks	10mg/kg, alternate days, 6wks	100mg/kg, alternate days, 18wks
1 day Post injection	18%	10%	12%	180%	17%	210%	13%	140%
2 day post	24%
3 day post	42%	64%
							4%	180%
	No GlcCer accumulation						No GlcCer accumulation

Uncertain Spans

location	transcription	uncertainty
D409V/D409V paper bullets / ii	`no GlcCer accumulation (=0
Stephens table	column ownership of 4%, 180%, No GlcCer accumulation cells	the right-edge two columns are partially clipped; cell-to-column alignment for the last 2-3 column slots is not fully visible on this capture.